Mitochondrial Aldehyde Dehydrogenase-2 Modulators and Methods of Use Thereof

ABSTRACT

The present invention provides compounds that function as modulators of mitochondrial aldehyde dehydrogenase-2 (ALDH2) activity; and pharmaceutical compositions comprising the compounds. The present invention provides therapeutic methods involving administering a subject compound, or a subject pharmaceutical composition. The present invention further provides assays for identifying agonists of ALDH2.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.13/214,937, filed Aug. 22, 2011, which is a continuation of U.S.application Ser. No. 12/044,870, filed Mar. 7, 2008, which claims thebenefit of U.S. Provisional Patent Application No. 60/905,963, filedMar. 8, 2007, the disclosures of each of which applications areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract AA011147awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Tissues deprived of blood and oxygen undergo ischemic necrosis orinfarction with possible irreversible organ damage. In somecircumstances, such as during surgery, interruption of blood flowresulting in ischemia of some organ is unavoidable. In addition, in thecase of solid tumors, it is desirable to interrupt the blood flow andactually induce ischemia. Once the flow of blood and oxygen is restoredto the organ or tissue (reperfusion), the organ does not immediatelyreturn to the normal preischemic state. For example, in the case of theischemic myocardium, reperfused postischemic non-necrotic myocardium ispoorly contractile and has reduced concentrations of high energynucleotides, depressed subcellular organelle function and membranedamage that resolves only slowly.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) is encoded in the nucleargenome and is transported into mitochondria. ALDH2 is a tetramericprotein composed of four identical subunits, each consisting of 500amino acid residues. This tetramer can be regarded as a dimer of dimers.The interface between monomers that form a dimer is different and moreextensive than the interface between the two dimers that form thetetramer. Each subunit is composed of three main domains: the catalyticdomain, the coenzyme or NAD⁺-binding domain, and the oligomerizationdomain.

LITERATURE

Larson et al. (2005) J. Biol. Chem. 280:30550; Li et al. (2006) J. Clin.Invest. 116:506; US Patent Publication No. 2005/0171043; PCT PublicationNo. WO 2005/057213.

SUMMARY OF THE INVENTION

The present invention provides compounds that function as modulators ofmitochondrial aldehyde dehydrogenase-2 (ALDH2) activity; andpharmaceutical compositions comprising the compounds. The presentinvention provides therapeutic methods involving administering a subjectcompound, or a subject pharmaceutical composition. The present inventionfurther provides assays for identifying agonists of ALDH2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B provide the amino acid sequence of human ALDH2 (SEQID NO:1) and the amino acid sequence of an E487K variant of human ALDH2,respectively.

FIG. 2 schematically depicts a fluorescent aldehyde dehydrogenaseenzymatic assay.

FIG. 3 depicts the effect of two exemplary ALDH2 agonists on enzymaticactivity of an E487K variant of human ALDH2.

FIG. 4 depicts the effect of two exemplary ALDH2 agonists on enzymaticactivity of an E487K variant of human ALDH2.

FIG. 5, panels a-c, depict the structure of exemplary ALDH2 agonists(FIG. 5, panel a); and results showing the specificity of exemplaryALDH2 agonists for ALDH2 (FIG. 5, panels b and c).

FIG. 6, panels a-d, depict the effect of an exemplary ALDH2 agonist inan ex vivo model of myocardial infarction.

FIG. 7 depicts the activity of various ALDH2 agonists on an E487Kvariant of human ALDH2.

FIG. 8 depicts inhibition of ALDH2 by an exemplary ALDH2 antagonist.

FIG. 9 depicts inhibition of ALDH2 by an exemplary ALDH2 antagonist.

FIG. 10 depicts inhibition of ALDH2 by an exemplary ALDH2 antagonist.

FIG. 11 depicts the effect of an exemplary ALDH2 agonist in an in vivomodel of myocardial infarction.

FIG. 12 depicts the effect of compounds BDDB, XO-43, and XO-44 on ALDH2activity.

DEFINITIONS

As used herein, the term “mitochondrial aldehyde dehydrogenase-2” or“ALDH2” refers to an enzyme that oxidizes an aldehyde (e.g., a xenogenicaldehyde, a biogenic aldehyde, or an aldehyde produced from a compoundthat is ingested, inhaled, or absorbed) to its corresponding acid in anNAD⁺-dependent reaction. For example, ALDH2 oxidizes aldehydes derivedfrom the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, or that are producedduring normal metabolism.

The term “ALDH2” encompasses ALDH2 from various species. Amino acidsequences of ALDH2 from various species are publicly available. Forexample, a human ALDH2 amino acid sequence is found under GenBankAccession Nos. AAH02967 and NP_(—)000681; a mouse ALDH2 amino acidsequence is found under GenBank Accession No. NP_(—)033786; and a ratALDH2 amino acid sequence is found under GenBank Accession No.NP_(—)115792. The term “ALDH2” as used herein also encompassesfragments, fusion proteins, and variants (e.g., variants having one ormore amino acid substitutions, addition, deletions, and/or insertions)that retain ALDH2 enzymatic activity. Specific enzymatically activeALDH2 variants, fragments, fusion proteins, and the like can be verifiedby adapting the methods described herein. An example of an ALDH2 variantis an ALDH2 polypeptide that comprises a Glu-to-Lys substitution atamino acid position 487 of human ALDH2, as depicted in FIG. 1B (aminoacid 504 of SEQ ID NO:2), or at a position corresponding to amino acid487 of human ALDH2. This mutation is referred to as the “E487Kmutation”; the “E487K variant”; or as the “Glu504Lys polymorphism”. See,e.g., Larson et al. (2005) J. Biol. Chem. 280:30550; and Li et al.(2006) J. Clin. Invest. 116:506. An ALDH2 variant retains at least about1% of the enzymatic activity of a corresponding wild-type ALDH2 enzyme.For example, the E487K variant retains at least about 1% of the activityof an enzyme comprising the amino acid sequence depicted in FIG. 1A (SEQID NO:1).

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Isolated compounds are at least about80%, at least about 90% pure, at least about 98% pure, or at least about99% pure, by weight. The present invention is meant to comprehenddiastereomers as well as their racemic and resolved, enantiomericallypure forms and pharmaceutically acceptable salts thereof.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient, in combination withanother agent, or alone in one or more doses, to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to a member or members of any mammalian ornon-mammalian species that may have a need for the pharmaceuticalmethods, compositions and treatments described herein. Subjects andpatients thus include, without limitation, primate (including humans),canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)),avian, and other subjects. Humans and non-human mammals havingcommercial importance (e.g., livestock and domesticated animals) are ofparticular interest.

“Mammal” refers to a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. a non-human primate, a murine (e.g.,a mouse, a rat), lagomorpha, etc. may be used for experimentalinvestigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes one and morethan one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and is free of contaminants that are capable of eliciting anundesirable response within the subject (e.g., the compound(s) in thepharmaceutical composition is pharmaceutical grade). Pharmaceuticalcompositions can be designed for administration to subjects or patientsin need thereof via a number of different routes of administrationincluding oral, buccal, rectal, parenteral, intraperitoneal,intradermal, intracheal and the like. In some embodiments thecomposition is suitable for administration by a transdermal route, usinga penetration enhancer other than dimethylsulfoxide (DMSO). In otherembodiments, the pharmaceutical compositions are suitable foradministration by a route other than transdermal administration. Apharmaceutical composition will in some embodiments include a subjectcompound and a pharmaceutically acceptable excipient. In someembodiments, a pharmaceutically acceptable excipient is other than DMSO.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

A “pharmaceutically acceptable ester” of a compound of the inventionmeans an ester that is pharmaceutically acceptable and that possessesthe desired pharmacological activity of the parent compound, andincludes, but is not limited to, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl estersof acidic groups, including, but not limited to, carboxylic acids,phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids andboronic acids.

A “pharmaceutically acceptable enol ether” of a compound of theinvention means an enol ether that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

“Pro-drugs” means any compound that releases an active parent drugaccording to one or more of the generic formulas shown below in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of one or more of the generic formulas shown below are preparedby modifying functional groups present in the compound of the genericformula in such a way that the modifications may be cleaved in vivo torelease the parent compound. Prodrugs include compounds of one or moreof the generic formulas shown below wherein a hydroxy, amino, orsulfhydryl group in one or more of the generic formulas shown below isbonded to any group that may be cleaved in vivo to regenerate the freehydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to esters (e.g., acetate, formate, andbenzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) ofhydroxy functional groups in compounds of one or more of the genericformulas shown below, and the like.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combinations thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Halogens of particular interest include chloro and bromogroups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature, or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

A subject compound may possess one or more asymmetric centers; suchcompounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amitochondrial aldehyde dehydrogenase-2 agonist” includes a plurality ofsuch agonists and reference to “the pharmaceutical composition” includesreference to one or more pharmaceutical compositions and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides compounds that function as modulators ofmitochondrial aldehyde dehydrogenase-2 (ALDH2) activity; andpharmaceutical compositions comprising the compounds. Agonists of ALDH2are useful for treating a variety of disorders, including, e.g.,conditions involving ischemic stress, chronic free-radical associateddiseases, acute free-radical associated diseases, insensitivity tonitroglycerin (e.g., in angina and heart failure), hypertension,diabetes, and osteoporosis. Agonists of ALDH2 are also useful forreducing the level in an individual of a compound such as ethanol,methanol, ethylene glycol monomethyl ether, polyvinyl chloride,xenogenic aldehydes, and biogenic aldehydes. Agonists of ALDH2 are alsouseful for reducing the level in an individual of a compound that, wheningested, absorbed, or inhaled, gives rise to an aldehyde substrate forALDH2. Antagonists of ALDH2 are useful for treating disorders such ascancer, where the ALDH2 antagonist is used as an adjuvant to a standardcancer therapy. Antagonists of ALDH2 are also useful for treatingalcoholism. Antagonists of ALDH2 are also useful for treating narcoticaddiction. The present invention provides therapeutic methods involvingadministering a subject compound, or a subject pharmaceuticalcomposition. The present invention further provides assays foridentifying agonists of ALDH2.

In some embodiments, individuals to be treated are humans. In someembodiments, a human to be treated according to a subject method is onethat has two “wild-type” ALDH2 alleles, e.g., the ALDH2 encoded by thetwo wild-type ALDH2 alleles has a glutamic acid at position 487, asdepicted in FIG. 1A. In other embodiments, a human to be treatedaccording to a subject method is one that has one or two “ALDH2*2”alleles, e.g., the ALDH2 encoded by one or both ALDH2 alleles comprisesa lysine as amino acid position 487, as depicted in FIG. 1B. The E487Kpolymorphism is a semidominant polymorphism, and results in an ALDH2tetramer that has significantly lower enzymatic activity than“wild-type” ALDH2. Thus, individuals who are heterozygous or homozygousfor the ALDH2*2 allele have much lower in vivo ALDH2 activity levelsthan individuals who are homozygous for the “wild-type” ALDH2 allele.Individuals who are heterozygous or homozygous for the ALDH2*2 alleleare expected to benefit from treatment with a subject ALDH2 agonist,because the level of ALDH2 activity in such individuals is particularlylow, and any increase of ALDH2 activity levels would be expected toprovide a therapeutic effect. Any increase in ALDH2 activity would bebeneficial in treating conditions such as ischemic disorders, inincreasing the responsiveness of such individuals to nitroglycerin,etc., as discussed in more detail below.

The use of ALDH2 variants, such as an E487K ALDH2 variant, in screeningmethods to identify ALDH2 activators (agonists) is also provided.Because the E487K ALDH2 variant has lower enzymatic activity than the“wild-type” ALDH2, the readout for agonist activity of a test compoundis more sensitive.

Modulators of Mitochondrial Aldehyde Dehydrogenase-2

The present invention provides compounds that function as modulators ofmitochondrial aldehyde dehydrogenase-2 (ALDH2) activity; andpharmaceutical compositions comprising the compounds. Modulators includeagonists (also referred to herein as “activators”) and antagonists (alsoreferred to herein as “inhibitors”).

In some embodiments, a compound that modulates ALDH2 activity modulatesa dehydrogenase activity of ALDH2, e.g., the compound modulatesdehydrogenase activity in oxidizing an aldehyde (e.g., a xenogenicaldehyde, a biogenic aldehyde, or an aldehyde produced from a compoundthat is ingested, inhaled, or absorbed) to the corresponding acid. Inother embodiments, a compound that modulates ALDH2 activity modulates anesterase activity of ALDH2. In other embodiments, a compound thatmodulates ALDH2 activity modulates a reductase activity of ALDH2. Forexample, ALDH2 can convert nitroglycerin to nitric oxide (NO) via itsreductase activity.

As noted above, in some embodiments, a compound that modulates ALDH2activity modulates a dehydrogenase activity of ALDH2, e.g., the compoundmodulates dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid.

A variety of compounds can give rise to aldehyde substrates for ALDH2.Non-limiting examples of compounds that can give rise to aldehydesubstrates for ALDH2 include ethanol; a variety of insecticides;industrial toxins such as vinyl chlorides (e.g., polyvinyl chloride);and pyruvate. For example, a compound is ingested, absorbed (e.g.,through the skin), or inhaled, by a mammal and is subsequently convertedin the mammal into an aldehyde substrate for ALDH2.

Biogenic aldehydes include aldehydes that are produced by a mammal,e.g., are produced metabolically by a mammal. Non-limiting examples ofbiogenic aldehydes include co-6 polyunsaturated fatty acids, such asmalondialdehyde (MDA); hexanal; acrolein; glyoxal; crotonaldehyde;trans-2-nonenal; 4-oxo-2-nonenal; and 4-hydroxy-2-nonenal (HNE) (seee.g., Ellis, Pharmacology & Therapeutics (2007) 115:13, Picklo andMontine (2007) J Alzheimers Dis. 12:185); 3-aminopropanal (3-AP), aproduct of polyamine oxidase; and aldehyde products of tyrosine, serineand threonine (see Wood et al, Brain Res (2006)1095; 190).

Xenogenic aldehydes include aldehydes ingested, absorbed, or inhaled bya mammal from source outside the mammal. Xenogenic aldehydes include,e.g., formaldehyde and glutaraldehyde (e.g., McGregor et al., Crit RevToxicol (2006) 36:821 and Pandey et al Hum Exp. Toxicol. (2000) 19:360);chloroacetaldehyde (see e.g., Richardson et al., Mutat. Research (2007)636:178); and reactive aldehydes present in cigarette smoke (see Simthet al., Inhal. Toxicol. (2006) 18:667).

Non-limiting examples of compounds that are substrates for mitochondrialALDH2 include 3,4-dihydroxypheylacetaldehyde (DOPAL); formaldehyde;acetaldehyde; propionaldehyde; n-butyraldehyde; capronaldehyde;heptaldehyde; pentaldehyde; octylaldehyde; decylaldehyd; retinaldehyde;3-hydroxybenzaldehyde; 2,5-dihydroxybenzaldehyde; phenylacetaldehyde;3-phenylpropionaldehyde (see, e.g., Want et al. (2002) Drug Metabolismand Disposition 30:69); cinnamoyl and hydrocinnamoyl aldehydes and theirderivative aldehydes (e.g. p-nitrocinnamaldehyde,p-(dimethylamino)cinnamaldehyde, hydrocinnamaldehyde,α-phenylpropionaldehyde); benzaldehyde and its derivative aldehydes(e.g. 2,4-dinitro-benzaldehyde, o-nitro-benzaldehyde,p-nitro-benzaldehyde, p-methyl-benzaldehyde, m-methyl-benzaldehyde,p-methoxy-benzaldehyde, p-(dimethylamino)-benzaldehyde,m-methoxy-benzaldehyde, m-hydroxy-benzaldehyde,3,4-dimethoxy-benzaldehyde, o-methoxy-benzaldehyde); naphthaldehyde andits derivative aldehydes (e.g. 5-bromo-1-naphthaldehyde,5-nitro-1-naphthaldehyde, 6[O—(CH₂)₅—COOH]-2-naphthaldehyde,6-(dimethylamino)-2-naphthaldehyde); coumarin-4-carboxaldehyde and itsderivative aldehydes (e.g. 7-acetoxy-coumarin-4-carboxaldehyde,7-(dimethylamino)-coumarin-4-carboxaldehyde,7-methoxy-coumarin-4-carboxaldehyde,6,7-dimethoxy-coumarin-4-carboxaldehyde); quinoline,quinolinonecarboxaldehyde, and their derivative aldehydes (e.g.quinoline-3-carboxaldehyde,7-(dimethylamino)-2-quinolinone-4-carboxaldehyde,quinoline-4-carboxaldehyde, 6-methoxy-2-quinolinone-4-carboxaldehyde);phenanthrene-9-carboxaldehyde; indole-3-aldehyde,indole-3-actaldehyde;5-methoxyindole-3-carboxaldehyde; 3-pyridinecarboxaldehyde;fluorene-2-carboxaldehyde (see, e.g., Klyosov, (1996) Biochemstry35:4457); 4-hydroxynonenal; malondialdehyde;3,4-dihydroxyphenylacetaldehyde; and 5-hydroxylindole-3-acetaldehyde.See, also, e.g., Williams et al. (2005) Anal. Chem. 77:3383; Marchittiet al. (2007) Pharmacol. Rev. 59:125; and Hoffman and Maser (2007) DrugMetab. Rev. 39:87.

ALDH2 Agonists

The present invention provides ALDH2 agonists (also referred to as“activators”); and pharmaceutical compositions comprising ALDH2agonists. Agonists of ALDH2 are useful for treating a variety ofdisorders, including, e.g., conditions involving ischemic stress,chronic free-radical associated diseases, acute free-radical associateddiseases, insensitivity to nitroglycerin (e.g., in angina and heartfailure), hypertension, diabetes, and osteoporosis. Agonists are alsouseful in the detoxification of alcohol abuse, methanol poisoning,ethylene glycol monomethyl ether poisoning, and poisoning due to otherxenogenic or biogenic aldehyde compounds.

An ALDH2 agonist increases an enzymatic activity of an ALDH2 polypeptideby at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 100% (or two-fold), at least about 2.5-fold, at leastabout 5-fold, at least about 10-fold, at least about 15-fold, at leastabout 20-fold, at least about 25-fold, or at least about 50-fold, orgreater than 50-fold, when compared to the enzymatic activity of theALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases a dehydrogenase activity(e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH2 polypeptide by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, an ALDH2 agonist increases the esterase activity ofan ALDH2 polypeptide by at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the esteraseactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases the reductase activityof an ALDH2 polypeptide by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 100% (or two-fold), at leastabout 2.5-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, or atleast about 50-fold, or greater than 50-fold, when compared to thereductase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, an ALDH2 agonist increases an enzymatic activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517 ofSEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the enzymaticactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases a dehydrogenase activity(e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, an ALDH2 agonist increases an esterase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517 ofSEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the esteraseactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases a reductase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517 ofSEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the reductaseactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases an enzymatic activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:2 (depicted in FIG. 1B), or as set forth in amino acids 18-517 ofSEQ ID NO:2, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the enzymaticactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases a dehydrogenase activity(e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forth in aminoacids 18-517 of SEQ ID NO:2, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, an ALDH2 agonist increases an esterase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:2 (depicted in FIG. 1B), or as set forth in amino acids 18-517 ofSEQ ID NO:2, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the esteraseactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, an ALDH2 agonist increases a reductase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:2 (depicted in FIG. 1B), or as set forth in amino acids 18-517 ofSEQ ID NO:2, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the reductaseactivity of the ALDH2 polypeptide in the absence of the agonist.

In some embodiments, ALDH2 agonists are specific for (e.g., selectivefor) ALDH2, e.g., a subject ALDH2 agonist increases an enzymaticactivity of an ALDH2 enzyme, but does not substantially increase thesame enzymatic activity of cytosolic aldehyde dehydrogenase-1 (ALDH1),e.g., a subject ALDH2 agonist increases an enzymatic activity of anALDH1 enzyme, if at all, by less than about 5%, less than about 2%, orless than about 1%, when used at a concentration that increases the sameenzymatic activity of an ALDH2 enzyme by at least about 5% or more. Insome embodiments, a subject ALDH2 agonist does not substantiallyincrease the enzymatic activity of alcohol dehydrogenase (ADH), e.g., asubject ALDH2 agonist increases the enzymatic activity of an ADH, if atall, by less than about 5%, less than about 2%, or less than about 1%,when used at a concentration that increases the enzymatic activity of anALDH2 enzyme by at least about 5% or more.

For example, in some embodiments, a subject ALDH2 agonist is specificfor (e.g., selective for) ALDH2, e.g., a subject ALDH2 agonist increasesdehydrogenase activity of an ALDH2 enzyme, but does not substantiallyincrease the dehydrogenase activity of cytosolic aldehydedehydrogenase-1 (ALDH1), e.g., a subject ALDH2 agonist increasesdehydrogenase activity of an ALDH1 enzyme, if at all, by less than about5%, less than about 2%, or less than about 1%, when used at aconcentration that increases dehydrogenase activity of an ALDH2 enzymeby at least about 5% or more. In some embodiments, a subject ALDH2agonist does not substantially increase dehydrogenase activity ofalcohol dehydrogenase (ADH), e.g., a subject ALDH2 agonist increases thedehydrogenase activity of an ADH, if at all, by less than about 5%, lessthan about 2%, or less than about 1%, when used at a concentration thatincreases the dehydrogenase activity of an ALDH2 enzyme by at leastabout 5% or more.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ of from about 1nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nMto about 15 nM, from about 15 nM to about 25 nM, from about 25 nM toabout 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about100 nM, from about 100 nM to about 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 450 nM, from about 450 nM to about500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, fromabout 75 μM to about 100 μM, from about 100 μM to about 250 μM, fromabout 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

For example, in some embodiments, a subject ALDH2 agonist has an EC₅₀ offrom about 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM,from about 10 nM to about 15 nM, from about 15 nM to about 25 nM, fromabout 25 nM to about 50 nM, from about 50 nM to about 75 nM, from about75 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 450 nM, from about 450 nM toabout 500 nM, from about 500 nM to about 750 nM, from about 750 nM toabout 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,from about 75 μM to about 100 μM, from about 100 μM to about 250 μM,from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM,for a dehydrogenase activity of mitochondrial ALDH2.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ for an ALDH2polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1(depicted in FIG. 1A), or as set forth in amino acids 18-517 of SEQ IDNO:1, of from about 1 nM to about 1 mM, e.g., from about 1 nM to about10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM,from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, fromabout 75 nM to about 100 nM, from about 100 nM to about 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 450 nM, fromabout 450 nM to about 500 nM, from about 500 nM to about 750 nM, fromabout 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, from about 75 μM to about 100 μM, from about 100 μM toabout 250 μM, from about 250 μM to about 500 μM, or from about 500 μM toabout 1 mM.

For example, in some embodiments, a subject ALDH2 agonist has an EC₅₀for dehydrogenase activity of an ALDH2 polypeptide comprising an aminoacid sequence set forth in SEQ ID NO:1 (depicted in FIG. 1A), or as setforth in amino acids 18-517 of SEQ ID NO:1, of from about 1 nM to about1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15nM, from about 15 nM to about 25 nM, from about 25 nM to about 50 nM,from about 50 nM to about 75 nM, from about 75 nM to about 100 nM, fromabout 100 nM to about 150 nM, from about 150 nM to about 200 nM, fromabout 200 nM to about 250 nM, from about 250 nM to about 300 nM, fromabout 300 nM to about 350 nM, from about 350 nM to about 400 nM, fromabout 400 nM to about 450 nM, from about 450 nM to about 500 nM, fromabout 500 nM to about 750 nM, from about 750 nM to about 1 μM, fromabout 1 μM to about 10 μM, from about 10 μM to about 25 μM, from about25 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μMto about 100 μM, from about 100 μM to about 250 μM, from about 250 μM toabout 500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ for an ALDH2polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2(depicted in FIG. 1B), or as set forth in amino acids 18-517 of SEQ IDNO:2, of from about 1 nM to about 1 mM, e.g., from about 1 nM to about10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM,from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, fromabout 75 nM to about 100 nM, from about 100 nM to about 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 450 nM, fromabout 450 nM to about 500 nM, from about 500 nM to about 750 nM, fromabout 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, from about 75 μM to about 100 μM, from about 100 μM toabout 250 μM, from about 250 μM to about 500 μM, or from about 500 μM toabout 1 mM.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ fordehydrogenase activity of an ALDH2 polypeptide comprising an amino acidsequence set forth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forthin amino acids 18-517 of SEQ ID NO:2, of from about 1 nM to about 1 mM,e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15 nM,from about 15 nM to about 25 nM, from about 25 nM to about 50 nM, fromabout 50 nM to about 75 nM, from about 75 nM to about 100 nM, from about100 nM to about 150 nM, from about 150 nM to about 200 nM, from about200 nM to about 250 nM, from about 250 nM to about 300 nM, from about300 nM to about 350 nM, from about 350 nM to about 400 nM, from about400 nM to about 450 nM, from about 450 nM to about 500 nM, from about500 nM to about 750 nM, from about 750 nM to about 1 μM, from about 1 μMto about 10 μM, from about 10 μM to about 25 μM, from about 25 μM toabout 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about100 μM, from about 100 μM to about 250 μM, from about 250 μM to about500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH2 agonist is an N-benzyl-benzamidecompound. In some embodiments, a subject ALDH2 agonist is a compound ofgeneric Formula I, as shown below:

where each of R₁, R₂, and R₃ is independently selected from H; a halo(e.g., bromo, fluoro, chloro, iodo); a substituted or unsubstitutedphenyl group; an aliphatic group, an alkyl group; a substituted alkylgroup; an alkenyl group; an alkynyl group; a substituted orunsubstituted cyclic group; a substituted or unsubstituted heterocyclicgroup; a substituted or unsubstituted aryl group; and a substituted orunsubstituted heteroaryl group;

where A is C or S and where a=1 when A=C; and where a=2 when A=S; and

where Ar₁ and Ar₂ are independently selected from a substituted arylgroup, an unsubstituted aryl group, a substituted heteroaryl group, andan unsubstituted heteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

For example, in some embodiments, Ar₁ and Ar₂ of Formula I areindependently:

where R₄-R₈ are each independently selected from H; a halo (e.g., bromo,fluoro, chloro, iodo); a substituted or unsubstituted phenyl group; analiphatic group, an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; asubstituted or unsubstituted heterocyclic group; a substituted orunsubstituted aryl group; and a substituted or unsubstituted heteroarylgroup. In other embodiments, Ar₁ and Ar₂ of Formula I are independentlya substituted or unsubstituted heterocyclic group, e.g., a substitutedor unsubstituted pyridine, a thiazole, an imidazole, a thiophene, aquinoline, an isoquinoline, or a furan group.

In some embodiments, a subject ALDH2 agonist is an N-benzyl-benzamidecompound. In some embodiments, a subject ALDH2 agonist is a compound ofgeneric Formula II, as shown below:

where X_(n) and X_(y) are each independently H, C, N, 0, or a halogen(e.g., F, Br, Cl, or I); where n is the integer 0 or 1; where y is theinteger 0 or 1;

where

(dotted line) is an optional bond; where z is the integer 0, 1, or 2;

where A is C or S, and where a=1 when A=C; and where a=2 when A=S;

where Ar is an unsubstituted or substituted aryl group, a substitutedheteroaryl group, or an unsubstituted heteroaryl group; and

where R₁ to R₆ is each independently selected from H; a halo (e.g.,bromo, fluoro, chloro, iodo); a substituted or unsubstituted phenylgroup; an aliphatic group, an alkyl group; a substituted alkyl group; analkenyl group; an alkynyl group; a substituted or unsubstituted cyclicgroup; a substituted or unsubstituted heterocyclic group; a substitutedor unsubstituted aryl group; and a substituted or unsubstitutedheteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

For example, in some embodiments, Ar of Formula II is:

where R₄-R₈ are each independently selected from H; a halo (e.g., bromo,fluoro, chloro, iodo); a substituted or unsubstituted phenyl group; analiphatic group, an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; asubstituted or unsubstituted heterocyclic group; a substituted orunsubstituted aryl group; and a substituted or unsubstituted heteroarylgroup. In some embodiments, R₄ and R₈ are methyl groups, and R₅, R₆, andR₇ are H. In some embodiments, R₄ and R₈ are halogen groups (e.g.,bromo, fluoro, chloro, iodo), and R₅, R₆, and R₇ are H.

In other embodiments, Ar of Formula II is a substituted or unsubstitutedheterocyclic group, e.g., a substituted or unsubstituted pyridine, athiazole, an imidazole, a thiophene, a quinoline, an isoquinoline, or afuran group. In some embodiments, Ar of Formula II is a substitutedpyridinyl group, e.g., a dihalogeno-substituted pyridinyl group.

In some embodiments, a subject ALDH2 agonist is a compound of genericformula Ia, as shown below:

where X_(n) and X_(y) are each independently H, C, N, 0, or a halogen(e.g., F, Br, Cl, or I);

where

(dotted line) is an optional bond;

where z is the integer 0, 1, or 2, with the provisos that: 1) z=0 whenX=halogen and is not a bond; and 2) when z=0, X═O,

is not a bond, and one or more oxygen atoms (X) are present, oxygen isattached to a methyl group;

where n is the integer 0 or 1;

where y is the integer 0 or 1;

where A=C or S, and where a=1 when A=C; and where a=2 when A=S;

where Ar is a phenyl or thiophene ring; wherein the Ar is optionallysubstituted at the position(s) ortho to the carbonyl or sulfonyl groupby one or more substituents independently selected from methyl, halo,trifluoromethyl, or phenyl; wherein Ar is optionally substituted by ahalogen meta or para to the carbonyl or sulfonyl group; and wherein,when Ar is a thiophene ring, the carbonyl or sulfonyl group is attachedto a thiophene ring at the 2 or 3 position;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH2 agonist has the structure ofCompound 1, as shown below.

Compound 1: (N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide)

In some embodiments, a subject ALDH2 agonist has the structure ofCompound 2, as shown below:

Compound 2: (N-(1,3-benzodioxol-5-ylmethyl)-2,6-difluorobenzamide)

In some embodiments, a subject ALDH2 agonist has the structure ofCompound 3, as shown below.

Compound 3: (N-(1,3-benzodioxol-5-ylmethyl)-2-bromobenzamide)

In some embodiments, one or more of Compound 1, Compound 2, and Compound3 is specifically excluded.

In some embodiments, a subject ALDH2 agonist has the structure ofCompound 4, as shown below.

Compound 4: (N-(1,3-benzodioxol-5-ylmethyl)-2-iodobenzamide)

In some embodiments, a subject ALDH2 agonist has the structure of one ofthe compounds designated XO-3, XO-4, XO-5, XO-9, XO-28, XO-29, XO-33,XO-36, XO-39, XO-12, XO-13, XO-6, XO-7, XO-8, XO-11, XO-22, XO-25, andXO-26, as shown below.

In some embodiments, a subject ALDH2 agonist has the structure ofCompound XO-43, as shown below:

In some embodiments, a subject ALDH2 agonist has the structure ofCompound XO-44, as shown below:

In some embodiments, a subject ALDH2 agonist has the structure ofCompound XO-45, as shown below:

In some embodiments, a subject ALDH2 agonist has the structure ofCompound XO-46, as shown below:

Whether a compound is an ALDH2 agonist can be readily ascertained.Assays for dehydrogenase activity of ALDH2 are known in the art, and anyknown assay can be used. Examples of dehydrogenase assays are found invarious publications, including, e.g., Sheikh et al. ((1997) J. Biol.Chem. 272:18817-18822); Vallari and Pietruszko (1984) J. Biol. Chem.259:4922; and Farres et al. ((1994) J. Biol. Chem. 269:13854-13860).

As an example of an assay for dehydrogenase activity, ALDH2 is assayedat 25° C. in 50 mM sodium pyrophosphate HCl buffer, pH 9.0, 100 mMsodium phosphate buffer, pH 7.4, or 50 mM sodium phosphate buffer, pH7.4, where the buffer includes NAD⁺ (e.g., 0.8 mM NAD⁺, or higher, e.g.,1 mM, 2 mM, or 5 mM NAD⁺) and an aldehyde substrate such as 14 μMpropionaldehyde. Reduction of NAD⁺ is monitored at 340 nm using aspectrophotometer, or by fluorescence increase using afluoromicrophotometer. Enzymatic activity can be assayed using astandard spectrophotometric method, e.g., by measuring a reductivereaction of the oxidized form of nicotinamide adenine dinucleotide(NAD⁺) to its reduced form, NADH, at 340 nm, as described in US2005/0171043; and WO 2005/057213, and as depicted schematically in FIG.2. In an exemplary assay, the reaction is carried out at 25° C. in 0.1NaPPi buffer, pH 9.5, 2.4 mM NAD⁺ and 10 mM acetaldehyde as thesubstrate. Enzymatic activity is measured by a reductive reaction ofNAD⁺ to NADH at 340 nm, as described in US 2005/0171043; and WO2005/057213. Alternatively, the production of NADH can be coupled withanother enzymatic reaction that consumes NADH and that provides for adetectable signal. An example of such an enzymatic reaction is adiaphorase-based reaction, which reduces resazurin to its oxidizedfluorescent compound resorufin, as described in US 2005/0171043; and WO2005/057213, and as depicted schematically in FIG. 2. Detection offluorescent resorufin at 590 nm provides amplified and more sensitivesignals for any change in ALDH2 enzymatic activity.

Whether a compound increases an esterase activity of ALDH2 can bedetermined using any known assay for esterase activity. For example,esterase activity of ALDH2 can be determined by monitoring the rate ofp-nitrophenol formation at 400 nm in 25 mM N,N-Bis(2-hydroxyethyl)-2-amino ethanesulfonic acid (BES) (pH 7.5) with 800 μMp-nitrophenyl acetate as the substrate at room temperature in theabsence or presence of added NAD⁺. A pH-dependent molar extinctioncoefficient of 16 mM⁻¹ cm⁻¹ at 400 nm for nitrophenol can be used. See,e.g., Larson et al. (2007) J. Biol. Chem. 282:12940). Esterase activityof ALDH2 can be determined by measuring the rate of p-nitrophenolformation at 400 nm in 50 mM Pipes (pH 7.4) with 1 mMp-nitrophenylacetate as the substrate. A molar extinction coefficient of18.3×10³ M⁻¹ cm⁻¹ at 400 nm for p-nitrophenolate can be used forcalculating its rate of formation. See, e.g., Ho et al. (2005)Biochemistry 44:8022).

Whether a compound increases a reductase activity of ALDH2 can bedetermined using any known assay for reductase activity. A reductaseactivity of ALDH2 can be determined by measuring the rate of1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate formation using a thinlayer chromatography (TLC) or liquid scintillation spectrometry method,using a radioactively labeled substrate. For example, 0.1 mM or 1 mM GTN(glyceryl trinitrate) is incubated with the assay mixture (1 ml)containing 100 mM KPi (pH 7.5), 0.5 mM EDTA, 1 mM NADH, 1 mM NADPH inthe presence ALDH2. After incubation at 37° C. for about 10 minutes toabout 30 minutes, the reaction is stopped and GTN and its metabolitesare extracted with 3×4 ml ether and pooled, and the solvent isevaporated by a stream of nitrogen. The final volume is kept to lessthan 100 ml in ethanol for subsequent TLC separation and scintillationcounting. See, e.g., Zhang and Stamler (2002) Proc. Natl. Acad. Sci. USA99:8306.

Another method for determining whether a compound increases enzymaticactivity of an ALDH2 is described in more detail below, where the E487KALDH2 variant is used.

ALDH2 Antagonists

The present invention provides ALDH2 antagonists (also referred to as“ALDH2 inhibitors”), and pharmaceutical compositions comprising ALDH2antagonists. In some embodiments, ALDH2 antagonists are useful fortreating alcohol addiction. In other embodiments, ALDH2 antagonistsincrease the sensitivity of a cancerous cell to a cancerchemotherapeutic agent. Thus, in some embodiments, ALDH2 antagonists areuseful as adjuvants to standard cancer therapies, in the treatment ofcancer.

In some embodiments, a subject ALDH2 antagonist reduces an enzymaticactivity of an ALDH2 polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, compared to the enzymaticactivity of the ALDH2 polypeptide in the absence of the antagonist.

In some embodiments, a subject ALDH2 antagonist reduces a dehydrogenaseactivity of an ALDH2 polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, compared to the dehydrogenaseactivity of the ALDH2 polypeptide in the absence of the antagonist.

In some embodiments, a subject ALDH2 antagonist reduces an esteraseactivity of an ALDH2 polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, compared to the esteraseactivity of the ALDH2 polypeptide in the absence of the antagonist.

In some embodiments, a subject ALDH2 antagonist reduces a reductaseactivity of an ALDH2 polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, compared to the reductaseactivity of the ALDH2 polypeptide in the absence of the antagonist.

In some embodiments, an ALDH2 antagonist reduces an enzymatic activityof an ALDH2 polypeptide comprising an amino acid sequence set forth inSEQ ID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517of SEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, when compared to the enzymatic activity of the ALDH2polypeptide in the absence of the antagonist.

In some embodiments, an ALDH2 antagonist reduces a dehydrogenaseactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%, when compared to the dehydrogenaseactivity of the ALDH2 polypeptide in the absence of the antagonist.

In some embodiments, an ALDH2 antagonist reduces an esterase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517 ofSEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, when compared to the esterase activity of the ALDH2polypeptide in the absence of the antagonist.

In some embodiments, an ALDH2 antagonist reduces a reductase activity ofan ALDH2 polypeptide comprising an amino acid sequence set forth in SEQID NO:1 (depicted in FIG. 1A), or as set forth in amino acids 18-517 ofSEQ ID NO:1, by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, when compared to the reductase activity of the ALDH2polypeptide in the absence of the antagonist.

In some embodiments, a subject ALDH2 antagonist is specific for (e.g.,selective for) ALDH2, e.g., a subject ALDH2 antagonist reduces theenzymatic activity of an ALDH2 enzyme, but does not substantially reducethe enzymatic activity of cytosolic aldehyde dehydrogenase-1 (ALDH1),e.g., a subject ALDH2 antagonist reduces the enzymatic activity of anALDH1 enzyme, if at all, by less than about 10%, less than about 5%,less than about 2%, or less than about 1%, when used at a concentrationthat reduces the enzymatic activity of an ALDH2 enzyme by at least about10% or more. In some embodiments, a subject ALDH2 antagonist does notsubstantially reduce the enzymatic activity of alcohol dehydrogenase(ADH), e.g., a subject ALDH2 antagonist reduces the enzymatic activityof an ADH, if at all, by less than about 10%, less than about 5%, lessthan about 2%, or less than about 1%, when used at a concentration thatreduces the enzymatic activity of an ALDH2 enzyme by at least about 10%or more.

For example, in some embodiments, a subject ALDH2 antagonist is specificfor (e.g., selective for) ALDH2, e.g., a subject ALDH2 antagonistreduces a dehydrogenase activity of an ALDH2 enzyme, but does notsubstantially reduce the dehydrogenase activity of cytosolic aldehydedehydrogenase-1 (ALDH1), e.g., a subject ALDH2 antagonist reduces thedehydrogenase activity of an ALDH1 enzyme, if at all, by less than about10%, less than about 5%, less than about 2%, or less than about 1%, whenused at a concentration that reduces the dehydrogenase activity of anALDH2 enzyme by at least about 10% or more. In some embodiments, asubject ALDH2 antagonist does not substantially reduce the dehydrogenaseactivity of alcohol dehydrogenase (ADH), e.g., a subject ALDH2antagonist reduces the dehydrogenase activity of an ADH, if at all, byless than about 10%, less than about 5%, less than about 2%, or lessthan about 1%, when used at a concentration that reduces thedehydrogenase activity of an ALDH2 enzyme by at least about 10% or more.

In some embodiments, a subject ALDH2 antagonist has an IC₅₀ of less than50 μM, e.g., a subject ALDH2 antagonist has an IC₅₀ of from about 50 μMto about 5 nm, or less than 5 nM. For example, in some embodiments, asubject ALDH2 antagonist has an IC₅₀ of from about 50 μM to about 25 μM,from about 25 μM to about 10 μM, from about 10 μM to about 5 μM, fromabout 5 μM to about 1 μM, from about 1 μM to about 500 nM, from about500 nM to about 400 nM, from about 400 nM to about 300 nM, from about300 nM to about 250 nM, from about 250 nM to about 200 nM, from about200 nM to about 150 nM, from about 150 nM to about 100 nM, from about100 nM to about 50 nM, from about 50 nM to about 30 nM, from about 30 nMto about 25 nM, from about 25 nM to about 20 nM, from about 20 nM toabout 15 nM, from about 15 nM to about 10 nM, from about 10 nM to about5 nM, or less than about 5 nM.

For example, in some embodiments, a subject ALDH2 antagonist has an IC₅₀of less than 50 μM, e.g., a subject ALDH2 antagonist has an IC₅₀ of fromabout 50 μM to about 5 nm, or less than 5 nM. For example, in someembodiments, a subject ALDH2 antagonist has an IC₅₀ of from about 50 μMto about 25 μM, from about 25 μM to about 10 μM, from about 10 μM toabout 5 μM, from about 5 μM to about 1 μM, from about 1 μM to about 500nM, from about 500 nM to about 400 nM, from about 400 nM to about 300nM, from about 300 nM to about 250 nM, from about 250 nM to about 200nM, from about 200 nM to about 150 nM, from about 150 nM to about 100nM, from about 100 nM to about 50 nM, from about 50 nM to about 30 nM,from about 30 nM to about 25 nM, from about 25 nM to about 20 nM, fromabout 20 nM to about 15 nM, from about 15 nM to about 10 nM, from about10 nM to about 5 nM, or less than about 5 nM, for a dehydrogenaseactivity of mitochondrial ALDH2.

In some embodiments, a subject ALDH2 agonist has an IC₅₀ for an ALDH2polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1(depicted in FIG. 1A), or as set forth in amino acids 18-517 of SEQ IDNO:1, of from about 50 μM to about 25 μM, from about 25 μM to about 10μM, from about 10 μM to about 5 μM, from about 5 μM to about 1 μM, fromabout 1 μM to about 500 nM, from about 500 nM to about 400 nM, fromabout 400 nM to about 300 nM, from about 300 nM to about 250 nM, fromabout 250 nM to about 200 nM, from about 200 nM to about 150 nM, fromabout 150 nM to about 100 nM, from about 100 nM to about 50 nM, fromabout 50 nM to about 30 nM, from about 30 nM to about 25 nM, from about25 nM to about 20 nM, from about 20 nM to about 15 nM, from about 15 nMto about 10 nM, from about 10 nM to about 5 nM, or less than about 5 nM.

In some embodiments, a subject ALDH2 agonist has an IC₅₀ for adehydrogenase activity of an ALDH2 polypeptide comprising an amino acidsequence set forth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forthin amino acids 18-517 of SEQ ID NO:1, of from about 50 μM to about 25μM, from about 25 μM to about 10 μM, from about 10 μM to about 5 μM,from about 5 μM to about 1 μM, from about 1 μM to about 500 nM, fromabout 500 nM to about 400 nM, from about 400 nM to about 300 nM, fromabout 300 nM to about 250 nM, from about 250 nM to about 200 nM, fromabout 200 nM to about 150 nM, from about 150 nM to about 100 nM, fromabout 100 nM to about 50 nM, from about 50 nM to about 30 nM, from about30 nM to about 25 nM, from about 25 nM to about 20 nM, from about 20 nMto about 15 nM, from about 15 nM to about 10 nM, from about 10 nM toabout 5 nM, or less than about 5 nM.

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula III, as shown below:

where Ar is an unsubstituted or substituted phenyl group;

where n=0, 1, 2, or 3;

where A=C or S, and where a=1 when A=C; and where a=2 when A=S; and

where R₁ and R₂ are independently H; a halo; a substituted orunsubstituted phenyl group; an amide, an aliphatic group, an alkylgroup; a substituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; a substituted orunsubstituted heterocyclic group; a substituted or unsubstituted arylgroup; and a substituted or unsubstituted heteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula

IV, as shown below:

where Ar is a substituted or unsubstituted phenyl or pyridyl group; and

where R₁ is selected from H; a halo (e.g., bromo, fluoro, chloro, iodo);a substituted or unsubstituted phenyl group; an amide, an aliphaticgroup, an alkyl group; a substituted alkyl group; an alkenyl group; analkynyl group; a substituted or unsubstituted cyclic group; asubstituted or unsubstituted heterocyclic group; a substituted orunsubstituted aryl group; and a substituted or unsubstituted heteroarylgroup;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, R₁ of Formula IV is selected from:

where each of R₂, R₃, R₄, and R₅ is independently selected from H; ahalo (e.g., bromo, fluoro, chloro, iodo); an amide; a substituted orunsubstituted phenyl group; an aliphatic group, an alkyl group; asubstituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; a substituted orunsubstituted heterocyclic group; a substituted or unsubstituted arylgroup; and a substituted or unsubstituted heteroaryl group.

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula V, as shown below:

R₁—(CH₂)_(n)—O—Ar—Z  (Formula V)

where n is an integer from 0 to 20 (e.g., 0, 1, 2, 2, 3, 4, 5-10, 10-15,or 15-20);

where Ar is an unsubstituted or substituted phenyl, naphthyl, orpyridyl;

where R₁ H; a halo (e.g., bromo, fluoro, chloro, iodo); an amide; asubstituted or unsubstituted phenyl group; an aliphatic group, an alkylgroup; a substituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; a substituted orunsubstituted heterocyclic group; a substituted or unsubstituted arylgroup; and a substituted or unsubstituted heteroaryl group;

where Z is H; a halo (e.g., bromo, fluoro, chloro, iodo); an amide; asubstituted or unsubstituted phenyl group; an aliphatic group, an alkylgroup; a substituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; a substituted orunsubstituted heterocyclic group; a substituted or unsubstituted arylgroup; and a substituted or unsubstituted heteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, Z of Formula V is selected from:

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula VI, as shown below:

where Ar is a substituted or unsubstituted phenyl or benzodioxaryl;

where R₁ and R₂ are independently H; a halo (e.g., bromo, fluoro,chloro, iodo); an amide; a substituted or unsubstituted phenyl group; analiphatic group, an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; asubstituted or unsubstituted heterocyclic group; a substituted orunsubstituted aryl group; and a substituted or unsubstituted heteroarylgroup;

where the dotted line is an optional benzene ring which may besubstituted or unsubstituted;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula VII, as shown below:

wherein:

Ar=substituted or unsubstituted aryl group;

Z=substituted or unsubstituted heterocyclic group;

Y═C, O, N, or S;

A=C or S, wherein when A=C, n=1, and wherein when A=S, n=2; and

m=an integer from 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 5-10, 10-15, or15-20),

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH2 antagonist is a compound of genericFormula VIIa, as shown below:

wherein:

Y═C, O, N, or S;

A=C or S, wherein when A=C, n=1, and wherein when A=S, n=2;

m=0, 1, 2, or 3;

each of R₁, R₂, R₃, and R₄ is independently selected from H, halogen,alkyl group, substituted alkyl group, alkenyl group, alkynyl group,hydroxyl, —CF₃, —OCF₃, —NO₂, substituted or unsubstituted amine,substituted or unsubstituted amide, substituted or unsubstituted cyclicgroup, substituted or unsubstituted heterocyclic group, substituted orunsubstituted aryl group, or substituted or unsubstituted heteroarylgroup; and

X₁ and X₂ are each independently C, N, O, or S, wherein a is 0, 1, 2, 3,or 4, and wherein b is 0, 1, 2, 3, or 4,

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH2 antagonist is Compound 5 (referredto in FIG. 9 as Compound 62923), as shown below:

In some embodiments, a subject ALDH2 antagonist is Compound 6 (referredto in FIG. 9 as Compound 46072), as shown below:

In some embodiments, a subject ALDH2 antagonist is Compound 7 (referredto in FIG. 10 as Compound 32208), as shown below:

In some embodiments, a subject ALDH2 antagonist is selected fromCompounds 8-18, as shown below:

Whether a compound is an ALDH2 antagonist can be readily ascertained.Assays for ALDH2 are known in the art, and any known assay can be used.Examples of assays are found in various publications, including, e.g.,Sheikh et al. ((1997) J. Biol. Chem. 272:18817-18822) and Farres et al.((1994) J. Biol. Chem. 269:13854-13860). For example, ALDH2 is assayedat 25° C. in 50 mM sodium pyrophosphate HCl buffer, pH 9.0, 100 mMsodium phosphate buffer, pH 7.4, or 50 mM sodium phosphate buffer, pH7.4, where the buffer includes NAD⁺ (e.g., 0.8 mM NAD⁺, or higher, e.g.,1 mM, 2 mM, or 5 mM NAD⁺) and a substrate such as 14 μM propionaldehyde.Reduction of NAD⁺ is monitored at 340 nm using a spectrophotometer, orby fluorescence increase using a fluoromicrophotometer. Enzymaticactivity can be assayed using a standard spectrophotometric method,e.g., by measuring a reductive reaction of the oxidized form ofnicotinamide adenine dinucleotide (NAD⁺) to its reduced form, NADH, at340 nm, as described in US 2005/0171043; and WO 2005/057213. In anexemplary assay, the reaction is carried out at 25° C. in 0.1 NaPPibuffer, pH 9.5, 2.4 mM NAD⁺ and 10 mM acetaldehyde as the substrate.Enzymatic activity is measured by a reductive reaction of NAD⁺ to NADHat 340 nm, as described in US 2005/0171043; and WO 2005/057213.Alternatively, the production of NADH can be coupled with anotherenzymatic reaction that consumes NADH and that provides for a detectablesignal. An example of such an enzymatic reaction is a diaphorase-basedreaction, which reduces resazurin to its oxidized fluorescent compoundresorufin, as described in US 2005/0171043; and WO 2005/057213.Detection of fluorescent resorufin at 590 nm provides amplified and moresensitive signals for any change in ALDH2 enzymatic activity.

Pharmaceutical Compositions, Dosages, Routes of Administration

The present invention provides pharmaceutical compositions comprising asubject

ALDH2 agonist. The present invention provides pharmaceuticalcompositions comprising a subject ALDH2 antagonist. The terms “ALDH2agonist” and “ALDH2 antagonist” are referred to collectively herein as“ALDH2 activity modulator” or “active agent.” A subject ALDH2 activitymodulator is formulated with one or more pharmaceutically acceptableexcipients. A wide variety of pharmaceutically acceptable excipients areknown in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy,” 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public

In the subject methods, a subject ALDH2 activity modulator may beadministered to the host using any convenient means capable of resultingin the desired reduction in autoimmune disease. Thus, a subject ALDH2activity modulator can be incorporated into a variety of formulationsfor therapeutic administration. More particularly, a subject ALDH2activity modulator can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, a subject active agent may beadministered in the form of their pharmaceutically acceptable salts, ora subject active agent may be used alone or in appropriate association,as well as in combination, with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

For oral preparations, a subject active agent can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

A subject active agent can be formulated into preparations for injectionby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

A subject active agent can be utilized in aerosol formulation to beadministered via inhalation. A subject active agent can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject active agent can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. An active agent can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycol monomethyl ethers, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the subject active agent. Similarly, unit dosageforms for injection or intravenous administration may comprise a subjectactive agent in a composition as a solution in sterile water, normalsaline or another pharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectactive agent calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject active agent dependon the particular compound employed and the effect to be achieved, andthe pharmacodynamics associated with each compound in the host.

A subject active agent can be formulated for administration byinjection. Typically, injectable compositions are prepared as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection may also be prepared.The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles.

In some embodiments, a subject active agent is delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of active agent can be accomplished usingany of a variety of refillable, pump systems. Pumps provide consistent,controlled release over time. In some embodiments, the agent is in aliquid formulation in a drug-impermeable reservoir, and is delivered ina continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the invention may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present invention.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT publishedapplication no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and5,728,396)). Exemplary osmotically-driven devices suitable for use inthe invention include, but are not necessarily limited to, thosedescribed in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426;3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202;4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850;4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692;5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted infra, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, an active agent is delivered using an implantabledrug delivery system, e.g., a system that is programmable to provide foradministration of a subject active agent. Exemplary programmable,implantable systems include implantable infusion pumps. Exemplaryimplantable infusion pumps, or devices useful in connection with suchpumps, are described in, for example, U.S. Pat. Nos. 4,350,155;5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704;6,464,687; 6,475,180; and 6,512,954. A further exemplary device that canbe adapted for the present invention is the Synchromed infusion pump(Medtronic).

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

Dosages and Dosing

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus, for example, oral dosages may be about ten times the injectiondose. Higher doses may be used for localized routes of delivery.

For example, a subject ALDH2 activity modulator can be administered inan amount of from about 1 mg to about 1000 mg per dose, e.g., from about1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg toabout 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100mg, from about 100 mg to about 125 mg, from about 125 mg to about 150mg, from about 150 mg to about 175 mg, from about 175 mg to about 200mg, from about 200 mg to about 225 mg, from about 225 mg to about 250mg, from about 250 mg to about 300 mg, from about 300 mg to about 350mg, from about 350 mg to about 400 mg, from about 400 mg to about 450mg, from about 450 mg to about 500 mg, from about 500 mg to about 750mg, or from about 750 mg to about 1000 mg per dose.

An exemplary dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is in some embodiments one whichprovides up to about 1 μg to about 1,000 μg or about 10,000 μg ofsubject compound in a blood sample taken from the individual beingtreated, about 24 hours after administration of the compound to theindividual.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound (s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

In some embodiments, multiple doses of a subject compound areadministered. The frequency of administration of a subject compound canvary depending on any of a variety of factors, e.g., severity of thesymptoms, etc. For example, in some embodiments, a subject compound isadministered once per month, twice per month, three times per month,every other week (qow), once per week (qw), twice per week (biw), threetimes per week (tiw), four times per week, five times per week, sixtimes per week, every other day (qod), daily (qd), twice a day (qid), orthree times a day (tid). As discussed above, in some embodiments, asubject compound is administered continuously.

The duration of administration of a subject compound, e.g., the periodof time over which a subject compound is administered, can vary,depending on any of a variety of factors, e.g., patient response, etc.For example, a subject compound can be administered over a period oftime ranging from about one day to about one week, from about two weeksto about four weeks, from about one month to about two months, fromabout two months to about four months, from about four months to aboutsix months, from about six months to about eight months, from abouteight months to about 1 year, from about 1 year to about 2 years, orfrom about 2 years to about 4 years, or more. In some embodiments, asubject compound is administered for the lifetime of the individual.

Routes of Administration

A subject ALDH2 activity modulator is administered to an individualusing any available method and route suitable for drug delivery,including in vivo and ex vivo methods, as well as systemic and localizedroutes of administration. Administration can be acute (e.g., of shortduration, e.g., a single administration, administration for one day toone week), or chronic (e.g., of long duration, e.g., administration forlonger than one week, e.g., administration over a period of time of fromabout 2 weeks to about one month, from about one month to about 3months, from about 3 months to about 6 months, from about 6 months toabout 1 year, or longer than one year).

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, transdermal, sublingual, topical application, intravenous,rectal, nasal, oral, and other enteral and parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the agent and/or the desired effect. Thecompound can be administered in a single dose or in multiple doses.

An active agent can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Treatment Methods

The present invention provides various treatment methods, generallyinvolving administering to an individual in need thereof an effectiveamount of a subject agonist, or an effective amount of a subjectantagonist. A subject ALDH2 agonist is suitable for treating a varietyof disorders, including, e.g., conditions involving ischemic stress,chronic free-radical associated diseases, acute free-radical associateddiseases, insensitivity to nitroglycerin (e.g., in angina and heartfailure), hypertension, diabetes, and osteoporosis. A subject ALDH2antagonist is suitable for sensitizing a cancerous cell to a cancerchemotherapeutic agent or other standard cancer therapy; for treatingalcohol (e.g., ethanol; ethyl alcohol) addiction; and for treatingnarcotic addiction.

Methods of Treating Conditions Involving Ischemic Stress

The present invention provides methods for treating conditions involvingischemic stress, including prophylactic methods, in an individual, themethods generally involving administering to an individual in needthereof an effective amount of a subject ALDH2 agonist. Conditionsinvolving ischemic stress include ischemic conditions, ischemic events,conditions that can give rise to ischemia, and conditions that resultfrom an ischemic event. Conditions involving ischemic stress that areamenable to treatment with a subject method include ischemia that resultfrom any condition or event, including, but not limited to, myocardialinfarct (e.g., acute myocardial infarction), cardiac surgery, braintrauma, cerebrovascular disease, stroke, spinal cord injury,subarachnoid hemorrhage, major surgery in which ischemia to variety oforgans occur, organ transplantation, limb ischemia (e.g., resulting fromType 1 or Type 2 diabetes), and the like.

In some embodiments, the agent is administered before a predicted oranticipated ischemic event, e.g., from about 1 hour to about 1 weekbefore the ischemic event, e.g., from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 8hours, from about 8 hours to about 12 hours, from about 12 hours toabout 16 hours, from about 16 hours to about 24 hours, from about 24hours to about 36 hours, from about 36 hours to about 48 hours, fromabout 48 hours to about 72 hours, or from about 72 hours to about 1 weekpreceding the predicted or anticipated ischemic event.

Pretreatment with an active agent is desirable under certaincircumstances, for example, when a subject has already experienced astroke, when a subject is about to undergo cardiac surgery, etc. Forexample, a patient who has already experienced a stroke will have anincreased probability of experiencing a second stroke. Subjects who aresusceptible to transient ischemic attacks also have an increased risk ofa stroke. Subjects who suffer a subarachnoid hemorrhage may experiencefurther ischemic events induced by vasospasms that constrict the bloodvessels. Subjects who experience trauma to organs such as the brain arealso susceptible to an ischemic event. Subjects undergoing surgery overan extended period of time are also susceptible to an ischemic event.The above situations exemplify circumstances when a subject wouldbenefit from pretreatment with a subject ALDH2 agonist.

In some embodiments, a subject ALDH2 agonist is administered after anischemic event. For example, a subject ALDH2 agonist is effective inreducing the adverse effects of an ischemic event such as cardiacischemia, reperfusion injury, cerebrovascular disease, acute myocardialinfarction, subarachnoid hemorrhage, and trauma. In some embodiments, asubject ALDH2 agonist is administered within 1 minute to within 15hours, e.g., from about 1 minute to about 5 minutes, from about 5minutes to about 10 minutes, from about 10 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes toabout 60 minutes, from about 60 minutes to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 8 hours, from about8 hours to about 12 hours, or from about 12 hours to about 15 hours,following the ischemic event. In some embodiments, an increasedconcentration of a subject ALDH2 agonist is maintained in the plasma forat least several hours to several days following the ischemic event.

For example, in some embodiments, a subject ALDH2 agonist isadministered to an individual who has suffered an acute myocardialinfarction (AMI) within 1 minute to within 15 hours, e.g., from about 1minute to about 5 minutes, from about 5 minutes to about 10 minutes,from about 10 minutes to about 15 minutes, from about 15 minutes toabout 30 minutes, from about 30 minutes to about 60 minutes, from about60 minutes to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 8 hours, from about 8 hours to about 12 hours, orfrom about 12 hours to about 15 hours, following the AMI.

Methods of Treating Chronic and Acute Free-Radical Associated Diseases

The present invention provides methods for treating acute and chronicfree-radical associated diseases in an individual, the methods generallyinvolving administering to an individual in need thereof an effectiveamount of a subject ALDH2 agonist.

Acute Free-Radical Associated Disorders

The present invention provides methods for treating acute free-radicalassociated diseases in an individual, the methods generally involvingadministering to an individual in need thereof an effective amount of asubject ALDH2 agonist. Acute free radical-associated disorders that areamenable to treatment with a subject method include seizures (Patel etal. (2001) Journal of Neurochemistry 79:1065-1069); skin damageresulting from UV exposure, and photodamage of skin (e.g., “sunburn”)(Aldini et al. (2007) Chem Res Toxicol. 20(3):416-23); acute thermalskin burn injury (Pintaudi et al. (2000) Free Radic Res. 33(2):139-46);and tissue hyperoxia (e.g., hyperoxia-induced chronic lung disease; andbronchopulmonary dysplasia) (Xu et al. (2006) Am J Physiol Lung Cell MolPhysiol. 291(5):L966-75).

The present invention provides methods for treating sunburn in anindividual, the methods generally involving administering to anindividual in need thereof an effective amount of a subject ALDH2agonist. In some embodiments, a subject method for treating sunburncomprises topically applying a formulation comprising a subject ALDH2agonist to an area of the skin affected by sunburn.

The present invention provides methods for treating a seizure in anindividual, the methods generally involving administering to anindividual in need thereof an effective amount of a subject ALDH2agonist. In some embodiments, a subject ALDH2 agonist is administeredafter a seizure has occurred, e.g., within from about 1 minute to about5 minutes, from about 5 minutes to about 15 minutes, from about 15minutes to about 30 minutes, from about 30 minutes to about 1 hour, orfrom about 1 hour to about 4 hours following a seizure. In otherembodiments, a subject ALDH2 agonist is administered prophylactically,e.g., a subject ALDH2 agonist is administered to an individual who hasexperienced a seizure in the past, to reduce the likelihood that anotherseizure will occur. In some embodiments, an effective amount of asubject ALDH2 agonist is an amount that is effective to reduce at leastone of the severity of a seizure, the frequency of seizures, and theduration of a seizure.

Chronic Free-Radical Associated Diseases

The present invention provides methods for treating chronic free-radicalassociated diseases in an individual, the methods generally involvingadministering to an individual in need thereof an effective amount of asubject ALDH2 agonist. Chronic free radical-associated disorders thatare amenable to treatment with a subject method includeneurodegenerative diseases such as Parkinson's Disease and Alzheimer'sDisease (Burke et al. (2003) Neurol. Dis. 2(2):143; and Ohta and Ohsawa(2006) J. Alzheimer's Disease 9(2):155); amyotrophic lateral sclerosis(ALS); cancer such as esophageal cancer (Chen et al. (2006) Int J Cancer2119(12):2827-31); upper aerodigestive tract cancer (Hashibe et al.(2006) Cancer Epidemiol Biomarkers Prev. 15(4):696-703); head and necksquamous cell carcinoma (Hashimoto et al. (2006) Tumour Biol.27(6):334-8; Yokoyama et al. (2005) Alcohol. 35(3):175-85);cardiovascular diseases such as atherosclerosis (Narita et al. (2003)Ultrasound in Medicine and Biology 29(10):1415-1419); and the like. Insome embodiments, a chronic free radical-associated disease is treatedby chronic (e.g., daily) treatment with a subject ALDH2 agonist.

The present invention provides a method for treating Alzheimer's Disease(AD) in an individual suffering from AD, the method generally involvingadministering to the individual an effective amount of a subject ALDH2agonist. In some embodiments, an “effective amount” of a subject ALDH2agonist is an amount that is effective to at least slow the decline incognitive function in the individual. In some embodiments, an “effectiveamount” of a subject ALDH2 agonist is an amount that is effective toimprove memory in the individual being treated. In some embodiments, asubject ALDH2 agonist is administered to the individual systemically,over a period of time of from about 3 months to about 6 months, fromabout 6 months to about 1 year, or more than 1 year.

The present invention provides a method for treating Parkinson's Diseasein an individual, the method generally involving administering to theindividual an effective amount of a subject ALDH2 agonist. In someembodiments, an “effective amount” of a subject ALDH2 agonist is anamount that is effective to ameliorate one or more symptoms ofParkinson's Disease. In some embodiments, an “effective amount” of asubject ALDH2 agonist is an amount that is effective to slow theprogress of the disease. In some embodiments, a subject ALDH2 agonist isadministered to the individual systemically, over a period of time offrom about 3 months to about 6 months, from about 6 months to about 1year, or more than 1 year.

Methods of Treating Heart Conditions

The present invention provides methods of treating disorders such asangina, heart failure, insensitivity to nitroglycerin in angina andheart failure (Li et al. (2006) J. Clin. Invest. 116:506-511),hypertension (Asselin et al. (2006) Free Radical Biol. and Med. 41:97),and heart disease. The methods generally involve administering to anindividual in need thereof an effective amount of a subject ALDH2agonist.

In some embodiments, a subject ALDH2 agonist is administered to anindividual in conjunction with nitroglycerin treatment. The subjectALDH2 agonist and the nitroglycerin can be administered by the sameroute of administration (e.g., oral, sublingual, transdermal,translingual, etc.). In the alternative, subject ALDH2 agonist and thenitroglycerin can be administered by different routes of administration.For example, in some embodiments, nitroglycerin is administeredsublingually, translingually, transdermally, or orally; and a subjectALDH2 agonist is administered via a different route of administration(e.g., intravenous, intramuscular, etc.). The ALDH2 agonist can beadministered before, during, or after administration of thenitroglycerin.

An effective amount of a subject ALDH2 agonist is an amount that, whenadministered in combination therapy with nitroglycerin, is effective toreduce angina by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%, or more,within a period of time of from about 1 minute to about 2 minutes, fromabout 2 minutes to about 3 minutes, from about 3 minutes to about 4minutes, from about 4 minutes to about 5 minutes, or from about 5minutes to about 10 minutes, following administration of the ALDH2agonist. In some embodiments, a subject ALDH2 agonist and nitroglycerinare administered substantially simultaneously, e.g., within about twominutes, within about 1 minute, or within about 30 seconds of oneanother. The term “combination therapy with nitroglycerin” encompassesadministration of a subject ALDH2 agonist substantially simultaneouslywith nitroglycerin; administration of a subject ALDH2 agonist beforeadministration of nitroglycerin; administration of a subject ALDH2agonist after administration of nitroglycerin; etc.

In some embodiments, an effective amount of a subject ALDH2 agonist isan amount that is effective to treat hypertension, e.g., to reduce oneor more symptoms or indications of hypertension in an individual. Forexample, in some embodiments, an effective amount of a subject ALDH2agonist is an amount that is effective to reduce blood pressure in theindividual by at least about 5%, at least about 10%, at least about 15%,at least about 20%, or at least about 25%, or more, or to bring theblood pressure of the individual to within a normal range.

In some embodiments, an effective amount of a subject ALDH2 agonist isan amount that is effective to treat heart disease, e.g., to reduce oneor more symptoms or indications of heart disease in an individual.Whether a given ALDH2 agonist is effective to treat heart disease can bedetermined using standard methods of assessing heart function, e.g.,electrocardiogram, angiogram, and the like.

Methods of Detoxification

The present invention provides methods of reducing the levels of a toxiccompound in an individual, the methods generally involving administeringto an individual in need thereof an effective amount of a subject ALDH2agonist. The present invention provides methods of treating a disorderassociated with or resulting from a toxic level of a compound (e.g., axenogenic aldehyde; a biogenic aldehyde; or a compound that, wheningested, absorbed, or inhaled, gives rise to an aldehyde substrate forALDH2), the methods generally involving administering to an individualin need thereof an effective amount of a subject ALDH2 agonist, wherethe level of the compound in the individual is reduced to a non-toxiclevel.

Toxic compounds whose levels can be reduced in an individual using asubject method include, but are not limited to, ethanol, methanol,ethylene glycol monomethyl ether, xenogenic aldehydes, biogenicaldehydes, and an aldehyde produced by in vivo metabolism of a compoundthat is ingested, absorbed, or inhaled. A subject ALDH2 agonist isadministered in an amount that is effective, when administered in one ormore doses, to reduce a toxic level of a compound such as ethanol,methanol, ethylene glycol monomethyl ether, xenogenic aldehydes,biogenic aldehydes, or an aldehyde produced by in vivo metabolism of acompound that is ingested, absorbed, or inhaled. In some embodiments,the aldehyde is acetaldehyde.

As an example, a subject ALDH2 agonist is administered to an individualfollowing excessive alcohol (e.g., ethanol) consumption; and toxiclevels of alcohol or aldehyde (e.g., an aldehyde that is a metabolicproduct of ethanol) in the individual are reduced by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or at least about 90%, or more, compared to the alcohol or aldehydelevels in the individual before treatment with the ALDH2 agonist. Insome embodiments, a subject ALDH2 agonist is administered in an amountthat is effective to reduce a toxic alcohol or aldehyde level by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, or more, within from about 5minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to 1 hour, from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 6hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH2 agonist. In some embodiments, a subjectALDH2 agonist is administered in an amount that is effective to reduce atoxic alcohol or aldehyde level to a non-toxic level within from about 5minutes to about 15 minutes, from about 15 minutes to about 30 minutes,from about 30 minutes to 1 hour, from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 6hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH2 agonist.

As an example, a subject ALDH2 agonist is administered to an individualfollowing excessive alcohol (e.g., ethanol) consumption; and levels ofacetaldehyde in the individual are reduced by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, or more, compared to the alcohol or aldehyde levels inthe individual before treatment with the ALDH2 agonist. In someembodiments, a subject ALDH2 agonist is administered in an amount thatis effective to reduce an acetaldehyde level by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, or more, within from about 5 minutes to about 15minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH2 agonist.

The present invention provides methods of reducing aldehyde toxicity,the methods generally involving administering an effective amount of asubject ALDH2 agonist. In some embodiments, an effective amount of anALDH2 agonist is an amount that is effective to reduce one or moresymptoms of aldehyde toxicity. For example, in some embodiments, aneffective amount of an ALDH2 agonist is an amount that is effective toreduce one or more symptoms of excess ethanol consumption, where suchsymptoms include, e.g., headache, dehydration, fatigue, nausea,vomiting, diarrhea, weakness, anxiety, irritability, photophobia,phonophobia, etc.

As an example, a subject ALDH2 agonist is administered to an individualhaving a toxic level of an aldehyde (e.g., following excessive ethanolconsumption); and toxic levels of an aldehyde in the individual arereduced by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or at least about 90%, or more, comparedto the level of the aldehyde in the individual before treatment with theALDH2 agonist. In some embodiments, a subject ALDH2 agonist isadministered in an amount that is effective to reduce a toxic aldehydelevel by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%, or more, within fromabout 5 minutes to about 15 minutes, from about 15 minutes to about 30minutes, from about 30 minutes to 1 hour, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH2 agonist. In some embodiments, a subjectALDH2 agonist is administered in an amount that is effective to reduce atoxic aldehyde level to a non-toxic level within from about 5 minutes toabout 15 minutes, from about 15 minutes to about 30 minutes, from about30 minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH2 agonist.

In some embodiments, a subject ALDH2 agonist reduces the level of bothethanol and an aldehyde, e.g., following excessive ethanol consumption,as described above.

As another example, a subject ALDH2 agonist is administered to anindividual having toxic levels of methanol or ethylene glycol monomethylether; and the toxic level of methanol or ethylene glycol monomethylether is reduced by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, or more,compared to the methanol or ethylene glycol monomethyl ether level inthe individual before treatment with the ALDH2 agonist. In someembodiments, a subject ALDH2 agonist is administered in an amount thatis effective to reduce a toxic methanol or ethylene glycol monomethylether level by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%, or more,within from about 5 minutes to about 15 minutes, from about 15 minutesto about 30 minutes, from about 30 minutes to 1 hour, from about 1 hourto about 2 hours, from about 2 hours to about 4 hours, from about 4hours to about 6 hours, or from about 6 hours to about 8 hours, or more,following administration of the ALDH2 agonist. In some embodiments, asubject ALDH2 agonist is administered in an amount that is effective toreduce a toxic methanol or ethylene glycol monomethyl ether level to anon-toxic level within from about 5 minutes to about 15 minutes, fromabout 15 minutes to about 30 minutes, from about 30 minutes to 1 hour,from about 1 hour to about 2 hours, from about 2 hours to about 4 hours,from about 4 hours to about 6 hours, or from about 6 hours to about 8hours, or more, following administration of the ALDH2 agonist.

As another example, a subject ALDH2 agonist is administered to anindividual exhibiting drug toxicity, e.g., a toxic level of an aldehydefollowing ingestion, absorption, or inhalation of a drug (e.g., apharmaceutical compound, an illicit drug, etc.). In some embodiments,the aldehyde is produced following ingestion, absorption, or inhalationof a drug, by metabolism of the drug in the body. The toxic level ofaldehyde is reduced by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, or more,compared to the level of the aldehyde in the individual before treatmentwith the ALDH2 agonist. In some embodiments, a subject ALDH2 agonist isadministered in an amount that is effective to reduce a toxic aldehydelevel by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%, or more, within fromabout 5 minutes to about 15 minutes, from about 15 minutes to about 30minutes, from about 30 minutes to 1 hour, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH2 agonist. In some embodiments, a subjectALDH2 agonist is administered in an amount that is effective to reduce atoxic aldehyde level to a non-toxic level within from about 5 minutes toabout 15 minutes, from about 15 minutes to about 30 minutes, from about30 minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH2 agonist.

Methods of Reducing Salsolinol Levels

The present invention provides methods of reducing salsolinol levels inan individual, the methods generally involving administering to theindividual an effective amount of a subject ALDH2 agonist. Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquionoline) is acondensation product of dopamine with acetaldehyde. Acetaldehyde is ametabolic product of ethanol. Plasma salsolinol levels are higher inalcoholic compared to non-alcoholics. Reduction of salsolinol levels isuseful in reducing alcohol addiction.

In some embodiments, an effective amount of a subject ALDH2 agonist isadministered to an individual in need thereof following excessivealcohol (e.g., ethanol) consumption; where the effective amount providesfor a reduction in the levels of salsolinol in the individual of atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, or more, compared to thesalsolinol levels in the individual before treatment with the ALDH2agonist. In some embodiments, an effective amount of a subject ALDH2agonist is administered to an individual in need thereof at any time(e.g., not necessarily following excessive alcohol consumption). In someembodiments, a subject ALDH2 agonist is administered in an amount thatis effective to reduce a salsolinol level by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, or more, within from about 5 minutes to about 15minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH2 agonist. In some of these embodiments, the individual is one whohas been diagnosed with alcoholism. Symptoms and diagnosis of alcoholismare described in, e.g., Enoch and Goldman (2002) American FamilyPhysician 65:441.

Methods of Treating Alcohol Addiction

The present invention provides methods of treating alcohol (ethanol)addiction in an individual. The methods generally involve administeringto an individual in need thereof an effective amount of a subject ALDH2antagonist.

A subject ALDH2 antagonist can be administered to an individual on aregular basis to treat alcohol addiction. For example, in someembodiments, a subject ALDH2 antagonist is administered to an individualin need thereof twice daily, daily, every other day, twice weekly, onceper week, or twice per month. A subject ALDH2 antagonist can beadministered in the form of a transdermal “patch” to treat alcoholaddiction.

“Treating alcohol addiction,” as used herein, includes achieving one ormore of the following: a reduction in the amount of alcohol consumed; areduction in the frequency at which alcohol is consumed; a reduction inthe craving for alcohol; and a reduction in one or more of the symptomsof excessive alcohol consumption. “Alcohol,” as used herein in thecontext of alcohol addiction, refers to ethanol, e.g., beveragescontaining 2%, 3%, 4% 5%, or more, by volume, ethanol, e.g., wine, beer,vodka, whiskey, and the like.

Methods of Treating Cancer

The present invention provides methods of treating cancer in anindividual. The methods generally involve administering to an individualin need thereof an effective amount of a subject ALDH2 antagonist inconjunction with a standard cancer therapy. Standard cancer therapiesinclude surgery (e.g., surgical removal of cancerous tissue), radiationtherapy, bone marrow transplantation, chemotherapeutic treatment,biological response modifier treatment, and certain combinations of theforegoing.

Radiation therapy includes, but is not limited to, x-rays or gamma raysthat are delivered from either an externally applied source such as abeam, or by implantation of small radioactive sources.

Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; therefore,compounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers suitable for use in connection with themethods of the invention include, but are not limited to, (1) inhibitorsof tyrosine kinase (RTK) activity; (2) inhibitors of serine/threoninekinase activity; (3) tumor-associated antigen antagonists, such asantibodies that bind specifically to a tumor antigen; (4) apoptosisreceptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8)colony-stimulating factors; (9) inhibitors of angiogenesis; and (10)antagonists of tumor necrosis factor.

A subject method is effective to reduce a tumor load by at least about5%, at least about 10%, at least about 20%, at least about 25%, at leastabout 50%, at least about 75%, at least about 85%, or at least about90%, up to total eradication of the tumor, when compared to a suitablecontrol. Thus, in these embodiments, an “effective amount” of a subjectALDH2 antagonist is an amount that, when administered in conjunctionwith a standard cancer therapy, is effective to reduce a tumor load byat least about 5%, at least about 10%, at least about 20%, at leastabout 25%, at least about 50%, at least about 75%, at least about 85%,or at least about 90%, up to total eradication of the tumor, whencompared to a suitable control. In an experimental animal system, asuitable control may be a genetically identical animal not treated withthe agent. In non-experimental systems, a suitable control may be thetumor load present before administering the agent. Other suitablecontrols may be a placebo control.

Whether a tumor load has been decreased can be determined using anyknown method, including, but not limited to, measuring solid tumor mass;counting the number of tumor cells using cytological assays;fluorescence-activated cell sorting (e.g., using antibody specific for atumor-associated antigen); computed tomography scanning, magneticresonance imaging, and/or x-ray imaging of the tumor to estimate and/ormonitor tumor size; measuring the amount of tumor-associated antigen ina biological sample, e.g., blood; and the like.

Other Disorders

A subject ALDH2 agonist can be administered to an individual in needthereof in the treatment of diabetes. A subject ALDH2 agonist can beadministered to an individual in need thereof in the treatment ofosteoporosis.

Diabetes

The present invention provides methods of treating diabetes, the methodsgenerally involving administering to an individual in need thereof aneffective amount of a subject ALDH2 agonist. In some embodiments, asubject method of treating diabetes provides for treatment of a disorderthat is a result of diabetes, e.g., diabetic nephrophathy, diabeticneuropathy, and the like.

In some embodiments, a subject ALDH2 agonist is administered in anamount that is effective to reduce a blood glucose level in anindividual, e.g., to reduce a blood glucose level in an individual by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, or at least about 50% whencompared to the blood glucose levels in the absence of treatment withthe agonist. In some embodiments, an effective amount of an ALDH2agonist is an amount that is effective to reduce blood glucose levels toa normal range. Normal fasting blood glucose levels are typically in therange of from about 70 mg/dL to about 110 mg/dL before a meal. Normalblood glucose levels 2 hours after a meal are usually less than about120 mg/dL. Normal blood glucose levels during an oral glucose tolerancetest (involving drinking a sugar solution containing about 75 g glucose;then measuring blood glucose levels at various times following drinkingthe sugar solution) include: less than 140 mg/dL 2 hours after drinkingthe sugar solution; and all readings between 0 and 2 hours afterdrinking the sugar solution less than 200 mg/dL. Blood glucose levelsare also sometimes expressed in mmol/L. Normal blood glucose levels aregenerally between about 4 mmol/L and 8 mmol/L. Normal blood glucoselevels are generally less than about 10 mmol/L 90 minutes after a meal;and from about 4 mmol/L to about 7 mmol/L before meals.

In some embodiments, a subject treatment method comprises administeringa subject ALDH2 agonist, and co-administering at least a secondtherapeutic agent (e.g., insulin) for the treatment of diabetes. Insulinthat is suitable for use herein includes, but is not limited to, regularinsulin, semilente, NPH, lente, protamine zinc insulin (PZI),ultralente, insuline glargine, insulin aspart, acylated insulin,monomeric insulin, superactive insulin, hepatoselective insulin, and anyother insulin analog or derivative, and mixtures of any of theforegoing. Insulin that is suitable for use herein includes, but is notlimited to, the insulin forms disclosed in U.S. Pat. Nos. 4,992,417;4,992,418; 5,474,978; 5,514,646; 5,504,188; 5,547,929; 5,650,486;5,693,609; 5,700,662; 5,747,642; 5,922,675; 5,952,297; and 6,034,054;and published PCT applications WO 00/121197; WO 09/010645; and WO90/12814. Insulin analogs include, but are not limited to, superactiveinsulin analogs, monomeric insulins, and hepatospecific insulin analogs.

Osteoporosis

The present invention provides methods of treating osteoporosis, themethods generally involving administering to an individual in needthereof an effective amount of a subject ALDH2 agonist. In someembodiments, an “effective amount” of an ALDH2 agonist is an amounteffective to increase bone density in the individual. In otherembodiments, an “effective amount” of an ALDH2 agonist is an amount thatis effective to reduce the rate of bone density loss.

Subjects Suitable for Treatment

Subjects suitable for treatment with a subject ALDH2 activity modulatorinclude individuals suffering from a condition described above;individuals at risk for developing a condition described above;individuals who have been treated for a condition described above withan agent other than a subject ALDH2 activity modulator, and who eitherfailed to respond to such treatment, or who initially responded to suchtreatment, but subsequently relapsed; individuals who are refractory totreatment with an agent other than a subject ALDH2 activity modulatorfor a condition described above; and individuals who cannot toleratetreatment with an agent other than a subject ALDH2 activity modulatorfor a condition described above.

Methods Involving Administering an ALDH2 Agonist

A subject treatment method involving administration of a subject ALDH2agonist is suitable for treating various conditions, as noted above,including disorders or conditions associated with or resulting fromoxidative stress; disorders or conditions associated with nitroglycerininsensitivity; disorders or conditions associated with toxic levels ofethyl alcohol, aldehyde, methanol, ethylene glycol monomethyl ether,biogenic or xenogenic aldehydes, etc.; and heart diseases andconditions, such as coronary artery disease, angina, etc. In someembodiments, the individual is a human who is homozygous for an ALDH2allele that encodes an ALDH2 having an amino acid sequence as depictedin FIG. 1A. In other embodiments, the individual is a human who carriesone or two ALDH2*2 alleles, where an ALDH2*2 allele encodes an ALDH2having the E487K variant as depicted in FIG. 1B.

Approximately 40% of the East Asian population carries the semidominantALDH2*2 allele. Such individuals can be characterized by a response toethanol consumption that includes one or more of facial flushing,nausea, and tachycardia. In addition, ALDH2*2 individuals are also lessresponsive to nitroglycerin treatment for such disorders as angina andcoronary artery disease. Individuals who are heterozygous or homozygousfor the ALDH2*2 allele are suitable for treatment with a subject methodinvolving administration of a subject ALDH2 agonist.

Methods of Treating Conditions Associated with Ischemic Stress

Subjects suitable for treatment with subject ALDH2 agonist includeindividuals who are scheduled to undergo cardiac surgery or who haveundergone cardiac surgery; individuals who have experienced a stroke;individuals who have suffered brain trauma; individuals who haveprolonged surgery; individuals who have suffered a myocardial infarct(e.g., acute myocardial infarction); individuals who suffer fromcerebrovascular disease; individuals who have spinal cord injury;individuals having a subarachnoid hemorrhage; and individuals who willbe subjected to organ transplantation. Subjects suitable for treatmentwith a subject ALDH2 agonist also include individuals having an ischemiclimb disorder, e.g., resulting from Type 1 or Type 2 diabetes.

Methods of Treating Acute Free-Radical Associated Diseases

Subjects suitable for treatment with subject ALDH2 agonist includeindividuals who are having or who have experienced a seizure;individuals having skin damage resulting from UV exposure; individualshaving photodamage of the skin; individuals having an acute thermal skinburn injury; and individuals suffering from tissue hyperoxia.

Methods of Treating Chronic Free-Radical Associated Diseases

Subjects suitable for treatment with subject ALDH2 agonist includeindividuals who have been diagnosed with Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, or otherneurodegenerative disease; individuals having atherosclerosis;individuals having esophageal cancer; individuals having head and necksquamous cell carcinoma; and individuals having upper aerodigestivetract cancer.

Methods of Treating Cardiac Conditions

Subjects suitable for treatment with a subject ALDH2 agonist includeindividuals having angina; individuals having heart failure; individualswho exhibit an insensitivity to nitroglycerin in the treatment of anginaor heart failure; individuals having hypertension; and individualshaving heart disease.

Detoxification Methods

Subjects suitable for treatment with a subject ALDH2 agonist includeindividuals who have toxic levels of an aldehyde, e.g., via ingestion ofa toxic compound, via inhalation of a toxic compound, via ingestion orinhalation of toxic levels of a compound, or via production of thealdehyde during normal metabolism. Such individuals include, but are notlimited to, individuals who have ingested or inhaled ethanol, methanol,ethylene glycol monomethyl ether, or other xenogenic or biogenicaldehyde compounds. For example, such individuals include individualswho have ingested or inhaled pesticides, fungicides, or other suchcompounds; individuals who have consumed excessive levels of ethanol;and the like.

Methods of Treating Alcohol Addiction

Subjects suitable for treatment with a subject ALDH2 antagonist includeindividuals who have alcohol addiction, including individuals who areconsidered to be alcoholics (e.g., an individual having a primary,chronic disease characterized by one or more of: impaired control overdrinking alcohol, preoccupation with the drug alcohol, use of alcoholdespite adverse consequences, and distortions in thinking followingconsumption of alcohol); individuals suffering from withdrawal symptomsfollowing cessation of alcohol consumption; individuals experiencingalcohol dependence (e.g., alcohol abuse combined with tolerance,withdrawal, and an uncontrollable urge to drink alcohol); and the like.

Methods of Treating Diabetes

Subjects suitable for treatment with a subject ALDH2 agonist includeindividuals having Type 1 or Type 2 diabetes. Subjects suitable fortreatment include individuals who have been diagnosed with Type 1diabetes mellitus, where such individuals include those having a fastingblood glucose level greater than about 126 mg/dL. Such individualsinclude those having blood glucose levels of greater than about 200mg/dL following a two-hour glucose tolerance test (75 g anhydrousglucose orally). Subjects suitable for treatment include individuals whohave been diagnosed with Type 2 diabetes; individuals who have not yetbeen diagnosed with Type 2 diabetes, but who are at risk of developingType 2 diabetes, e.g., individuals having a body mass index (weight inkilograms divided by height (in meters) squared) greater than 25, e.g.,individuals having a body mass index from about 25 to about 27, fromabout 27 to about 30, or greater than 30.

Methods of Treating Cancer

Subjects suitable for treatment with a subject ALDH2 antagonist for thetreatment of cancer, as described above, include individuals having asolid tumor. Solid tumors include, but are not limited to, histiocyticlymphoma, cancers of the brain, genitourinary tract, lymphatic system,stomach, larynx and lung, including lung adenocarcinoma and small celllung cancer.

Screening Assays

The present invention provides methods for identifying an ALDH2 agonist.The methods generally involve contacting a variant ALDH2 enzyme havingreduced enzymatic activity with a test compound, in the presence of asubstrate for the variant ALDH2 enzyme; and determining the effect, ifany, of the test compound on the enzymatic activity of the variant ALDH2enzyme.

A “variant ALDH2 enzyme having reduced enzymatic activity” is a variantALDH2 enzyme that has reduced enzymatic activity relative to an ALDH2enzyme comprising an amino acid sequence depicted in FIG. 1A (SEQ IDNO:1), or comprising amino acids 18-517 of the an amino acid sequencedepicted in FIG. 1A, e.g., the variant ALDH2 enzyme exhibits, in vitroor in vivo, less than about 90%, less than about 80%, less than about75%, less than about 70%, less than about 60%, less than about 50%, lessthan about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, or less than about 2%, of the enzymaticactivity exhibited by an ALDH2 enzyme comprising an amino acid sequencedepicted in FIG. 1A (SEQ ID NO:1), or comprising amino acids 18-517 ofthe an amino acid sequence depicted in FIG. 1A. In some embodiments, avariant ALDH2 enzyme comprises an amino acid sequence depicted in FIG.1B (SEQ ID NO:2), or amino acids 18-517 of the an amino acid sequencedepicted in FIG. 1B.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 10,000 daltons,e.g., a candidate agent may have a molecular weight of from about 50daltons to about 100 daltons, from about 100 daltons to about 150daltons, from about 150 daltons to about 200 daltons, from about 200daltons to about 500 daltons, from about 500 daltons to about 1000daltons, from about 1,000 daltons to about 2500 daltons, from about 2500daltons to about 5000 daltons, from about 5000 daltons to about 7500daltons, or from about 7500 daltons to about 10,000 daltons. Candidateagents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the invention include controls, where suitable controlsinclude a sample (e.g., a sample comprising the variant ALDH2 enzyme andsubstrate in the absence of the test agent). Generally a plurality ofassay mixtures is run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc., including agents that are used to facilitate optimalenzyme activity and/or reduce non-specific or background activity.Reagents that improve the efficiency of the assay, such as proteaseinhibitors, anti-microbial agents, etc. may be used. The components ofthe assay mixture are added in any order that provides for the requisiteactivity. Incubations are performed at any suitable temperature,typically between 4° C. and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 hour and 1 hour will besufficient.

A test compound of interest is a compound that increases the enzymaticactivity of the variant ALDH2 by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to theenzymatic activity of the ALDH2 polypeptide in the absence of the testcompound.

In some embodiments, a test compound of interest is a compound thatincreases the enzymatic activity of an ALDH2 polypeptide comprising anamino acid sequence set forth in SEQ ID NO:2 (depicted in FIG. 1B), oras set forth in amino acids 18-517 of SEQ ID NO:2, by at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 100%(or two-fold), at least about 2.5-fold, at least about 5-fold, at leastabout 10-fold, at least about 15-fold, at least about 20-fold, at leastabout 25-fold, or at least about 50-fold, or greater than 50-fold, whencompared to the enzymatic activity of the ALDH2 polypeptide in theabsence of the test compound.

In some embodiments, a test compound of interest is a compound that isspecific for ALDH2, e.g., the test compound increases the enzymaticactivity of a variant ALDH2 enzyme, but does not substantially increasethe enzymatic activity of cytosolic aldehyde dehydrogenase-1 (ALDH1),e.g., the test compound increases the enzymatic activity of an ALDH1enzyme, if at all, by less than about 5%, less than about 2%, or lessthan about 1%, when used at a concentration that increases the enzymaticactivity of a variant ALDH2 enzyme by at least about 5% or more. In someembodiments, a test agent of interest does not substantially increasethe enzymatic activity of alcohol dehydrogenase (ADH), e.g., a testagent of interest increases the enzymatic activity of an ADH, if at all,by less than about 5%, less than about 2%, or less than about 1%, whenused at a concentration that increases the enzymatic activity of avariant ALDH2 enzyme by at least about 5% or more.

In some embodiments, a test compound of interest has an EC₅₀ of fromabout 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM, fromabout 10 nM to about 15 nM, from about 15 nM to about 25 nM, from about25 nM to about 50 nM, from about 50 nM to about 75 nM, from about 75 nMto about 100 nM, from about 100 nM to about 150 nM, from about 150 nM toabout 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 450 nM, from about 450 nM toabout 500 nM, from about 500 nM to about 750 nM, from about 750 nM toabout 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,from about 75 μM to about 100 μM, from about 100 μM to about 250 μM,from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

A candidate agent is assessed for any cytotoxic activity it may exhibittoward the cell used in the assay, using well-known assays, such astrypan blue dye exclusion, an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay, and the like. Agents that do not exhibit cytotoxic activity areconsidered candidate agents.

In many embodiments, the screening method is carried out in vitro, in acell-free assay. In some embodiments, the in vitro cell-free assay willemploy a purified variant ALDH2, where “purified” refers to free ofcontaminants or any other undesired components. Purified variant ALDH2that is suitable for a subject screening method is at least about 50%pure, at least about 60% pure, at least about 70% pure, at least about75% pure, at least about 80% pure, at least about 85% pure, at leastabout 90% pure, at least about 95% pure, at least about 98% pure, atleast about 99% pure, or greater than 99% pure.

Purified variant ALDH2 will in some embodiments be stabilized byaddition of one or more stabilizing agents, to maintain enzymaticactivity. In some embodiments, a solution of purified variant ALDH2comprises an aqueous solution of mitochondrial AldDH2 and from about 10%to about 50% glycerol, e.g., from about 10% to about 15%, from about 15%to about 20%, from about 20% to about 25%, from about 25% to about 30%,from about 30% to about 35%, from about 35% to about 40%, from about 40%to about 45%, or from about 45% to about 50% glycerol. In someembodiments, a solution of mitochondrial AldDH2 further comprises one ormore of a chelating agent (e.g., EDTA or EGTA); salts such as NaCl,MgCl₂, KCl, and the like; buffers, such as a Tris buffer,phosphate-buffered saline, sodium pyrophosphate buffer, and the like;one or more protease inhibitors; and the like.

In some embodiments, the in vitro cell-free assay will employ arecombinant variant

ALDH2. Recombinant variant ALDH2 is readily prepared in a variety ofhost cells such as unicellular microorganisms, or cells of multicellularorganisms grown in in vitro culture as unicellular entities. Suitablehost cells include bacterial cells such as Escherichia coli; yeast cellssuch as Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Yarrowia lipolytica, Candida utilis,Schizosaccharomyces pombe, and the like; insect cells such as Drosophilamelanogaster cells; amphibian cells such as Xenopus cells; mammaliancells, such as CHO cells, 3T3 cells, and the like. In some embodiments,the in vitro cell-free assay will employ a human variant ALDH2, e.g., avariant ALDH2 enzyme comprising an amino acid sequence as set forth inamino acids 18-517 of the sequence depicted in FIG. 1B. In someembodiments, the in vitro cell-free assay will employ a variant ALDH2produced recombinantly in E. coli cells.

In some embodiments, the in vitro cell-free assay will employ a fusionprotein, comprising a variant ALDH2 fused in-frame to a fusion partner.In some embodiments, the fusion partner is attached to the aminoterminus of the variant ALDH2 polypeptide. In other embodiments, thefusion partner is attached to the carboxyl terminus of the variant ALDH2polypeptide. In other embodiments, the fusion partner is fused in-frameto the variant ALDH2 polypeptide at a location internal to the variantALDH2 polypeptide. Suitable fusion partners include immunological tagssuch as epitope tags, including, but not limited to, hemagglutinin,FLAG, and the like; proteins that provide for a detectable signal,including, but not limited to, fluorescent proteins, enzymes (e.g.,β-galactosidase, luciferase, horse radish peroxidase, etc.), and thelike; polypeptides that facilitate purification or isolation of thefusion protein, e.g., metal ion binding polypeptides such as 6His tags(e.g., ALDH2/6His), glutathione-S-transferase, and the like;polypeptides that provide for subcellular localization; and polypeptidesthat provide for secretion from a cell.

In some embodiments, the fusion partner is an epitope tag. In someembodiments, the fusion partner is a metal chelating peptide. In someembodiments, the metal chelating peptide is a histidine multimer, e.g.,(His)₆. In some embodiments, a (His)₆ multimer is fused to the aminoterminus of the variant ALDH2; in other embodiments, a (His)₆ multimeris fused to the carboxyl terminus of the variant ALDH2. The(His)₆-variant ALDH2 fusion protein is purified using any of a varietyof available nickel affinity columns (e.g. His-bind resin, Novagen).

Assays for ALDH2 are known in the art, and any known assay can be usedin a subject screening method. Examples of assays are found in variouspublications, including, e.g., Sheikh et al. ((1997) J. Biol. Chem.272:18817-18822) and Farres et al. ((1994) J. Biol. Chem.269:13854-13860). For example, ALDH2 enzymatic activity is assayed at25° C. in 50 mM sodium pyrophosphate HCl buffer, pH 9.0, 100 mM sodiumphosphate buffer, pH 7.4, or 50 mM sodium phosphate buffer, pH 7.4,where the buffer includes NAD⁺ (e.g., 0.8 mM NAD⁺, or higher, e.g., 1mM, 2 mM, or 5 mM NAD⁺) and a substrate such as 14 μM propionaldehyde.Reduction of NAD⁺ is monitored at 340 nm using a spectrophotometer, orby fluorescence increase using a fluoromicrophotometer.

ALDH2 enzymatic activity can be assayed using a standardspectrophotometric method, e.g., by measuring a reductive reaction ofthe oxidized form of nicotinamide adenine dinucleotide (NAD⁺) to itsreduced form, NADH, at 340 nm, as described in US 2005/0171043, or WO2005/057213. In an exemplary assay, the reaction is carried out at 25°C. in 0.1 NaPPi buffer, pH 9.5, 2.4 mM NAD⁺ and 10 mM acetaldehyde asthe substrate. Enzymatic activity is measured by a reductive reaction ofNAD⁺ to NADH at 340 nm, as described in US 2005/0171043, or WO2005/057213. Alternative, the production of NADH can be coupled withanother enzymatic reaction that consumes NADH and that provides for adetectable signal. An example of such an enzymatic reaction is adiaphorase-based reaction, which reduces resazurin to its oxidizedfluorescent compound resorufin, as described in US 2005/0171043, or WO2005/057213. Detection of fluorescent resorufin at 590 nm providesamplified and more sensitive signals for any change in ALDH2 enzymaticactivity.

As one non-limiting example, a 120 μl reaction mixture for ALDH2enzymatic activity comprises the following components:

43 μl 150 mM sodium pyrophosphate (NaPPi) buffer, pH 9.0;

30 μl 10 mM NAD⁺;

15 μl 80 mM acetaldehyde;

1 μl of resazurin (0.2 mg/ml in H₂O);

1 μl of diaphorase (1 unit, e.g., from Clostridium kluyveri);

2 μl of variant ALDH2 (e.g., 2 μl of variant ALDH2 at (0.5-2 μg/μl); and

28 μl of a solution comprising an agent to be tested, which agent hasbeen resuspended in an appropriate solvent (e.g., an aqueous solution,DMSO, and the like).

Fluorescent detection of the above-described reaction as described inTable 1:

TABLE 1 Excitation Emission Cutoff Channel 1 340 nm 445 nm 410 nmChannel 2 565 nm 590 nm 570 nm

This reaction can be carried out in a 96-well, a 384-well, a 1536-wellmicro-well plate, etc., or adapted to other screening formats.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Identification and Characterization of ALDH2 Agonists MethodsIn Vitro Screen for ALDH2 Agonists (Activators) and Antagonists(Inhibitors)

Compounds were screened using a method as depicted schematically in FIG.2. Essentially, the reaction is carried out at 25° C. in 0.1 NaPPibuffer, pH 9.5, 2.4 mM NAD⁺ and 10 mM acetaldehyde as the substrate.Enzymatic activity is measured by a reductive reaction of NAD⁺ to NADHat 340 nm. Alternatively, the production of NADH can be coupled withanother enzymatic reaction that consumes NADH and that provides for adetectable signal. An example of such an enzymatic reaction is adiaphorase-based reaction, which reduces resazurin to its oxidizedfluorescent compound resorufin.

For example, a 120 μl reaction mixture for ALDH2 enzymatic activitycomprises the following components:

43 μl 150 mM sodium pyrophosphate (NaPPi) buffer, pH 9.0;

30 μl 10 mM NAD⁺;

15 μl 80 mM acetaldehyde;

1 μl of resazurin (0.2 mg/ml in H₂O);

1 μl of diaphorase (1 unit, e.g., from Clostridium kluyveri);

2 μl of variant ALDH2 (e.g., 2 μl of variant ALDH2 at (0.5-2 μg/μl); and

28 μl of a solution comprising an agent to be tested, which agent hasbeen resuspended in an appropriate solvent (e.g., an aqueous solution,DMSO, and the like).

Fluorescent detection of the above-described reaction as described inTable 1:

TABLE 1 Excitation Emission Cutoff Channel 1 340 nm 445 nm 410 nmChannel 2 565 nm 590 nm 570 nm

Histidine-Tagged E487K ALDH2 Protein

A fusion protein that includes the E487K variant of human ALDH2 (seeamino acid sequence depicted in FIG. 1B) and a poly-histidine tag wassynthesized and used in screens for ALDH2 agonists. An expressionconstruct that encodes the E87K variant fused in-frame to apoly-histidine (His) tag was introduced into E. coli, and expression wasinduced by the addition of isopropyl-1-thio-3-D-galactoside (IPTG); andHis-tagged E487K ALDH2 variant was produced. The His-tagged E487K ALDH2variant was purified from E. coli extract using a metal ion affinitycolumn to bind the His-tagged E487K ALDH2 variant. His-tagged E487KALDH2 variant was eluted from the column and used in screening assays toidentify ALDH2 agonists.

The His-tagged E487 ALDH2 was derived from amino acids 18-517 as shownin FIG. 1B, with the His-tag fused to the N-terminus of the E487variant.

Ex Vivo Assay

Ex vivo Langendorff preparations of rat hearts were used as a model toassess the damage incurred by no-flow ischemia and reperfusion injury.This is an experimental model that mimics the clinical situation ofmyocardial infarction in patients. Rat hearts were excised andcannulated on a Langendorff apparatus via the aorta. Retrogradeperfusion was carried out using the standard oxygenated Kreb-Hensleitbuffer maintained at 37° C. All hearts were stabilized by an initial 5-to 10-minute perfusion period followed by delivery of differentcardioprotective agents or AldDH2 inhibitors for 10-30 minutes,depending on the reagent. Reagents used in some of the representativeexperiments including ethanol (50 mM), EPKC isozyme-selective activatorand inhibitor peptides (1 μM), cyanamide (5 mM) and nitroglycerin (2μM). Ischemia was then introduced by 25 minutes of no-flow followed by60 minutes of reperfusion.

The degree of ischemia/reperfusion damage was measured by twoindependent commonly accepted parameters. In one assay, a cross-sectionof heart slices were obtained immediately after reperfusion and stainedwith 2,3,5-triphenyl-tetrazolium chloride (TTC) for infarct sizemeasurement. In another assay, creatine phosphate kinase activity wasmeasured from reperfusate of each heart collected during reperfusion.The data indicated that the two methods yielded comparable results forthe assessment of cardiac damage. Homogenates from each heart was alsoobtained from a separate section of the identical sample and analyzedfor aldehyde dehydrogenase enzymatic activity. Enzymatic activity wasdetermined by a standard spectrophotometric method described above usingacetaldehyde as the substrate and NAD⁺ as a cofactor.

Results

The histidine-tagged E487K ALDH2 variant was used to screen for ALDH2activators. Of 63,000 compounds tested, 3 compounds were identified asALDH2 activators. The initial 3 compounds were as follows:

This compound is also referred to as Compound 1, and isN-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide.

This compound is also referred to as Compound 2, and isN-(1,3-benzodioxol-5-ylmethyl)-2,6-difluorobenzamide.

This compound is also referred to as Compound 3, and isN-(1,3-benzodioxol-5-ylmethyl)-2-bromobenzamide.

The activity of compounds 2 and 3 was tested against the E487K ALDH2variant. The results are depicted in FIG. 3. Compound 2 and Compound 3increased enzymatic activity of the E487K ALDH2 variant approximately300% and 600%, respectively, over control (without activator compound)activity. (FIG. 3: n=3 for both left and right panels.)

A 2-iodo variant of Compound 3 was synthesized. The variant isN-(1,3-benzodioxol-5-ylmethyl)-2-iodobenzamide. The activity of the2-iodo variant and Compound 1 was tested against the E487K ALDH2variant. The results are depicted in FIG. 4. Compound 1 and the 2-iodovariant of Compound 3 increased enzymatic activity of the E487K ALDH2variant approximately 900% and 700%, respectively, over control (withoutactivator compound) activity. (FIG. 4: n=3).

Specificity of ALDH2 Agonists

The activity of Compounds 1, 2, and 3 was tested against wild-type ALDH2(where “wild-type” ALDH2 has a glutamic acid at position 487, asdepicted in FIG. 1A), against the E487K ALDH2 variant, and againstALDH1A1. Compound structures are depicted in FIG. 5A. The results aredepicted in FIGS. 5B and 5C. In in vitro assays, activity of thewild-type ALDH2 enzyme is from 41% to 65%. Compounds 1-3 activated theE487K ALDH2 variant by from 300% to 900% at 10 μM to 20 μM. Activationof ALDH2 by benzodioxol benzamide compounds (e.g., Compounds 1-3)appeared to be selective: There was no significant effect of thesecompounds on the enzymatic activity of ADH1 or the closely relatedcytosolic form of aldehyde dehydrogenase 1 (ALDH1).

Activity of ALDH2 Agonists in Myocardial Infarction Model.

The effect of compound 1 was tested in an ex vivo model of myocardialinfarction, to determine whether Compound 1 could protect myocardiumfrom ischemia-reperfusion damage. The results are shown in FIGS. 6A-6D.As shown in FIG. 6A, infarct size was significantly reduced from57.7±5.4% to 41.6±4.1, p<0.05, n=7. FIG. 6B depicts images of heartsections exposed to IR injury in the presence and absence ofN-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (BDDB). Whitetissue is infarcted. FIG. 6C depicts CPK release. CPK release wassignificantly decreased from 5530±329 Units/Liter to 4396 Units/Liter *P<0.05, n=8). FIG. 6D depicts the increase in ALDH2 enzymatic activityin compound 1-treated hearts 3.3±0.30 to 2.5±0.11 mole NADH/min/mgprotein, **p<0.01, n=6).

Synthesis of Additional Compounds

Several additional compounds were synthesized. The structures of thesecompounds (referred to as XO-3, XO-4, XO-5, XO-6, XO-7, XO-8, XO-9,XO-11, XO-12, XO-13, XO-22, XO-25, XO-26, XO-28, XO-29, XO-33, XO-36,and XO-39) are depicted above.

The compounds referred to as XO-3, XO-4, XO-5, XO-6, XO-7, XO-8, XO-9,XO-11, XO-12, XO-13, XO-22, XO-25, XO-26, XO-28, XO-29, XO-33, XO-36,and XO-39 were synthesized by reacting an amine with an acid chloride ora sulfonic acid chloride in the presence of a base. In one case,3,4-difluoro-benzylamine was reacted with 2-bromo-benzoyl chloride inthe presence of N,N-diisopropylethylamine in dichloromethane to formcompound XO-4.

All compounds were confirmed by thin layer chromatography, ¹H-nuclearmagnetic resonance, and mass spectrometry.

FIG. 7 depicts the effect of these compounds (XO-3, XO-4, XO-5, XO-6,XO-7, XO-8, XO-9, XO-11, XO-12, XO-13, XO-22, XO-25, XO-26, XO-28,XO-29, XO-33, XO-36, and XO-39) on activity of the E487K ALDH2 variant.All compounds were used at 10 μM, and assays were carried out induplicate. (FIG. 7: n=3).

Example 2 Identification and Characterization of ALDH2 Inhibitors

Compounds were screened, as described above, but using the “wild-type”ALDH2 enzyme. Of 63,000 compounds screened, six inhibitors wereidentified. Three of the compounds, referred to in FIGS. 8, 9, and 10 as#062923, #046072, and #032208, respectively, were assayed for theireffect on ALDH2 enzymatic activity. The results are shown in FIGS. 8-10.As shown in FIG. 8, compound #062923 inhibited ALDH2 activity at an IC₅₀of 0.63 μM. (Note that for FIGS. 3, 4, and 8-10, the x-axis unit is: ODat 340 nm. For FIGS. 3, 4, and 7-10, the y-axis unit is: Time (inminutes). As shown in FIG. 9, compound #046072 inhibited ALDH2 activityat an IC₅₀ of 1.14 μM. (FIG. 9: n=3). As shown in FIG. 10, compound#032208 inhibited ALDH2 activity at an IC₅₀ of 1.62 μM. (FIG. 10: n=3).

The structures of additional compounds (referred to as Compounds 8-18,above) that were identified in the initial screen as ALDH2 inhibitorsare provided above.

Example 3 BDDB Protects Against Ischemia/Reperfusion Injury In Vivo

Male Wistar rats (250-300 g) were anesthetized by 3% isoflurane. Thesurgical procedures used for left anterior descending coronary artery(LAD) ligation are based on published protocols Animals were intubated,and ventilated with a Harvard rodent ventilator at a rate of 80 breathsper minute (5-15 mm Hg). Maintenance anesthesia was provided via 1%inhalational isoflurane, and body temperature was maintained at 37° C.using a rectal probe linked to a thermocoupled thermometer and anappropriate heating blanket. The heart was exposed by median sternotomy.Following a 20-minute period of stabilization, a ligature was placedaround the LAD coronary artery, close to its origin from the aorticroot. The ends of the ligature were passed through polyethylene tubingforming a small noose in which a syringe plunger was rested upon themyocardial surface. Coronary occlusion was achieved by pressing thetubing against the plunger while pulling on the ligature followed byclamping the tubing with a hemostat. Occlusion was determined byobservation of immediate pallor of the left ventricular free wall.Reflow was achieved by release of the ligature.

Following exposure of the heart and stabilization, BDDB was infuseddirectly into the left ventricle using a 30 G needle connected to acatheter and inserted through the apex of the heart into the leftventricle. Correct placement was verified by a small backflow of bloodinto the catheter as well as post-mortem dissection. The catheter wasattached to a syringe and BDDB was infused at a rate of 0.08 ml/min witha target volume of 0.16 ml and a final concentration of 8.5 mg/kg. Fiveminutes after BDDB treatment, the LAD was occluded. Control hearts wereinjected with the same volume of DMSO at the same flow rate. In all theexperiments, the 35-minute occlusion was followed by 60 minutes ofreperfusion.

At the end of reperfusion, hearts were excised and flushed with 0.9%saline. Infarct size analysis was carried by the same procedures asdescribed in the ex vivo experiments using TTC. 0.45 ml of the bloodsample were withdrawn into a 0.05 ml heparin-primed syringe 15 minuteand 30 minute after termination of the LAD occlusion time points via anapical punch of the left ventricle. Serum was separated bycentrifugation at 5000 g for 5 minutes on a tabletop centrifuge andcreatine phosphate kinase (CPK) values were determined.

The results are shown in FIG. 11. Ligation of the left anteriordescending (LA) coronary artery resulted in 43% infarction of the leftventricular free wall and a significant creatine phosphokinase (CPK)release into the blood (FIG. 11). Administration of 8.5 mg/kg BDDB intothe left ventricle five minutes before LAD ligation decreased the extentof myocardial infarction by about 60% and CPK release by about 57% (FIG.11 p<0.01). These results indicated that ALDH2 activation is sufficientto protect the heart from ischemia damage, in vivo.

Example 4 Activation of ALDH2 by Agonist Compounds XO43, XO44 and BDDB

BDDB derivative compounds, XO43 and XO44 as depicted in FIG. 12, weresynthesized and tested in ALDH2 enzymatic assays. Both compounds showedpotency in activating ALDH2*1 and ALDH2*2 recombinant enzyme in astandard assay using 0.1 sodium pyrophosphate buffer, pH 9.5, 2.4 mMNAD⁺ and 10 mM acetaldehyde as the substrate at 25° C. Enzymaticactivity was measured by a reductive reaction of NAD⁺ to NADH at 340 nm,as described in US 2005/0171043; and WO 2005/057213. The data arepresented in FIG. 12. XO43, XO44 and BDDB showed a dosage dependenteffect of ALDH2 activation in the range of 10-200 μM. The ALDH2*1 wildtype enzyme showed an increase of about 180% in activity and the ALDH2*2mutant enzyme showed and increase of >900% at the higher concentration.(FIG. 12: n=3 for both right and left panels.)

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-27. (canceled)
 28. A pharmaceutical composition comprising: (a) acompound of the formula:

wherein X is O or F, provided that when X is F, Ar is a substituted arylgroup;

(dotted line) is an optional bond; z is the integer 0, 1, or 2, providedthat: 1) z=0 when X═F and is not a bond; and 2) when z=0, X═O,

is not a bond, and one or more oxygen atoms (X) are present, then oxygenis attached to a methyl group; n is the integer 0 or 1; y is the integer0 or 1; A=C or S, and where a=1 when A=C; and where a=2 when A=S; and Aris a substituted aryl group, a substituted heteroaryl group, or anunsubstituted heteroaryl group; provided that when Ar is a substitutedphenyl ring, Ar is substituted at one or both position(s) ortho to thecarbonyl or sulfonyl group by one or more substituents independentlyselected from methyl, halo, trifluoromethyl, or phenyl; and optionallysubstituted by one halogen meta or para to the carbonyl or sulfonylgroup; or a pharmaceutically acceptable salt thereof, wherein thecompound increases enzymatic activity of an aldehyde dehydrogenase-2polypeptide by at least about 20%; and (b) a pharmaceutically acceptableexcipient, provided that if Ar is substituted at the position para tothe carbonyl or sulfonyl group, then Ar is also substituted at bothpositions ortho to the carbonyl or sulfonyl group, if Ar is substitutedat the positions ortho and meta to the carbonyl or sulfonyl group or ifAr is a substituted heteroaryl group or an unsubstituted heteroarylgroup, then X═O, n=1, y=1, z=1 and . . . is a bond, and if Ar issubstituted at both positions ortho to the carbonyl or sulfonyl groupand z=0, then X═O, n=1 and y=1.
 29. The pharmaceutical composition ofclaim 28, wherein A is C, and wherein Ar is a phenyl ring substituted ata position ortho to the carbonyl group.
 30. The pharmaceuticalcomposition of claim 29, wherein the substitution is a halogen.
 31. Thepharmaceutical composition of claim 28, wherein Ar is a substitutedphenyl, or a thiophene ring, wherein, when Ar is a thiophene ring, thecarbonyl or sulfonyl group is attached to the thiophene ring at the 2 or3 position.
 32. The pharmaceutical composition of claim 28, wherein Aris a substituted or unsubstituted heteroaryl group.
 33. Thepharmaceutical composition of claim 28, wherein Ar is a phenyl ringsubstituted at the position(s) ortho to the carbonyl or sulfonyl groupby one or more halogens, and wherein Ar is optionally substituted by onehalogen meta or para to the carbonyl or sulfonyl group.
 34. Thepharmaceutical composition of claim 28, wherein Ar is a phenyl ringsubstituted with one or more methyl groups.
 35. The pharmaceuticalcomposition of claim 28, wherein X is O.
 36. The pharmaceuticalcomposition of claim 28, wherein the compound has an EC₅₀ for increasingthe activity of an aldehyde dehydrogenase-2 polypeptide of from about 1nM to about 100 nM.
 37. The pharmaceutical composition of claim 28,wherein the pharmaceutically acceptable excipient is notdimethylsulfoxide.
 38. The pharmaceutical composition of claim 37,wherein the pharmaceutically acceptable excipient comprises saline,dextrose, or ethanol.
 39. The pharmaceutical composition of claim 37,wherein the compound has an EC₅₀ for increasing the activity of analdehyde dehydrogenase-2 polypeptide of from about 1 nM to about 10 μM.40. A method of treating an ischemic stress condition in an individualin need thereof, the method comprising administering to the individualan effective amount of the pharmaceutical composition of claim
 37. 41. Amethod of treating an acute or a chronic free-radical associated diseasein an individual in need thereof, the method comprising administering tothe individual an effective amount of the pharmaceutical composition ofclaim
 37. 42. A method of treating angina, the method comprisingco-administering to an individual in need thereof a nitroglycerincompound, and a pharmaceutical composition of claim 28 in combinedeffective amounts to treat the angina.
 43. A method of reducing a levelof a compound present at a toxic level in an individual to below thetoxic level, the method comprising administering to the individual aneffective amount of the composition of claim 37, wherein the compoundpresent at a toxic level is ethanol, methanol, ethylene glycolmonomethyl ether, a vinyl chloride, a xenogenic aldehyde, a biogenicaldehyde, or a compound that give rise to a biogenic aldehyde.
 44. Amethod of modulating enzymatic activity of an aldehyde dehydrogenase-2(ALDH-2) polypeptide, the method comprising contacting the ALDH-2polypeptide with a pharmaceutical composition of claim
 28. 45. Thepharmaceutical composition of claim 28, wherein when A is C and Ar is aphenyl ring substituted at a position ortho to the carbonyl group, thesubstitution is not F.
 46. The pharmaceutical composition of claim 28,wherein the compound is not 2,6-dichloro-N-(3-methoxybenzyl)benzamide or2,6-dichloro-N-(4-methoxybenzyl)benzamide.